MAKETA po korektœre 2003

ISSN 1335-8421 Acta Med Mart 2003, 3(1) 

ACTA MEDICA 

MARTINIANA 

Journal for Biomedical Sciences, 

Clinical Medicine and Nursing 

Contents 

3 

Relation between QT and RR intervals during Valsalva manoeuvre in young healthy 

women 

Kujaník ·tefan, Rakárová Martina 

10 

Chemical control of breathing in anaesthetized rabbits during hyperthermia and its recovery 

by body surface cooling 

Îila Ivan, Brozmanová Andrea, Javorka Kamil, Javorka Michal, âalkovská Andrea, 

Petrá‰ková Mária 

15 

NADPH: cytochrome P450 reductases of various species can be used in systems reconstituting 

drug-metabolizing CYP2E1 activity 

Belejová Marie, Anzenbacherová Eva, Zuber Roman, Anzenbacher Pavel 

19 

Arboviruses in Slovakia 

Eleãková E, Labuda, M, Rajãáni J 

29 

Prevalence of prothrombin mutation gene (G→A 20210) 

in thrombophilic patients 

Chudej Juraj, Koneãná Stanislava, PlameÀová Ivana, Ivanková Jela, Hudeãek Jan, Sta‰ko Ján, 

Pullmann Rudolf, Melu‰ Vladimír, Kubisz Peter 

32 

Central versus intraparenchymal arteries of kidney: Comparison of Doppler parameters 

under the physiological conditions in newborns 

Stavûl Miroslav, Kollarovszka Hana, Strechová Zuzana, Poláãek Hubert, Zibolen Mirko 

Published by the Jessenius Faculty of Medicine in Martin, 

Comenius University in Bratislava, Slovakia

A C T A M E D I C A M A R T I N I A N A 3 / 1 

Editor-in-Chief: 

Javorka, K., Martin, Slovakia 

International Editorial Board: 

Belej, K., Martin, Slovakia 

Buchanec, J., Martin, Slovakia 

Honzíková, N., Brno, Czech Republic 

Kliment, J., Martin, Slovakia 

Lehotsk˘, J., Martin, Slovakia 

Lichnovsk˘, V., Olomouc, Czech Republic 

Mare‰, J., Praha, Czech Republic 

Plank, L., Martin, Slovakia 

Stránsky, A., Martin, Slovakia 

Tatár, M., Martin, Slovakia 

˚wirska-Korczala, K., Zabrze-Katowice, Poland 

Editorial Office: 

Acta Medica Martiniana 

Jessenius Faculty of Medicine, Comenius University 

(Dept. of Physiology) 

Malá Hora 4 

037 54 Martin 

Slovakia 

Instructions for authors: http:|www.jfmed.uniba.sk (Acta Medica Martiniana) 

Tlaã: 

ProKonzult, s. r. o., závod NADAS, Vrútky 

© Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia, 2003

A C T A M E D I C A M A R T I N I A N A 3 / 1 3 

RELATION BETWEEN QT AND RR INTERVALS DURING VALSALVA 

MANOEUVRE IN YOUNG HEALTHY WOMEN 

·TEFAN KUJANÍK, MARTINA RAKÁROVÁ 

Department of Physiology, Medical Faculty of ·afárik University, Ko‰ice, Slovak Republic 

Abstract 

Objective: The aim of the study was to investigate the relation between QT interval and cardiac cycle length (RR interval) 

during 4 phases of Valsalva manoeuvre (VM) in young healthy women. 

Material and methods: The electrocardiogram of 28 healthy women (18-24 years) was registered during resting normal 

ventilation (RNV=control) and during 4 phases of VM (20 s at 40 mmHg = 5.33 kPa) in the daytime (9:00 to 17:00). 

The relation between QT and RR intervals compared to RNV was expressed by manually measured alterations of QT, RR, 

QTc intervals (paired t-test), and by the single correlation coefficient „r“. 

Results: All examined electrocardiographic parametres during RNV were within normal limits in all persons. In all 

phases of VM the heart rate, QT, QTc, and RR intervals were significantly different (p < 0.001) from RNV, normal QTc 

values (under 440 ms) were in 17.86-57.14% of subjects in individual phases. The maximum alterations of measured 

parameters were in the third phase, the minimum ones in the first phase. RR intervals sometimes altered very markedly 

from one cardiac cycle to the other one while the QT interval altered almost not distinctly. The independence between 

duration of QT and RR intervals was the most distinct in the phase 4. The simple correlation coefficient was 0.8948 

at rest (p < 0.01), 0.8196 (1 st phase, p < 0.01), 0.8169 (2 nd phase, p < 0.01), 0.8381 (3 rd phase, p < 0.01), 0.7506 (4 th 

phase, p < 0.01), 0.7855 (total VM, p < 0.01) and QT-RR relation was very individual. 

Conclusions: The QT interval duration is little altered during VM inspite of evident alterations in RR interval. The 

highest QT-RR correlation is in RNV, the lowest one during the 4 th phase of VM. The QT-RR relation is very individual. 

Key words: electrocardiography, QT interval, RR interval, Valsalva manoeuvre, healthy persons 

INTRODUCTION 

The length of the QT interval of electrocardiogram (electrical systole) is an indicator of the 

electrical stability of the heart. The QT interval is considered to be dependent on the heart rate 

(HR), its duration decreases with increasing HR. In attempt to remove its dependence on the HR, 

many formulae for calculation of the corrected (independent on HR) QT interval (QTc) were proposed 

(1, 2). They are dependent on QT-RR relation and have some limitations. Therefore alterations 

in QTc interval may reflect different changes in duration of QT or RR. 

The laws of the relation between QT and RR intervals are not clear yet. It can change under 

different conditions, such as reflex cardiovascular reactions, under influence of many drugs, etc., 

when the rate dependence of QT interval is not firmly expressed. Within a wide range of RR intervals, 

the QT duration is altering only a little (3, 4, 5). Some respiratory manoeuvres (voluntary 

hyperventilation, hypoxic-hypercapnic ventilation, Valsalva manoeuvre) are able to alter the 

dependence of QT interval on the HR, expressed by regression lines, (6) and to influence the tone 

of cardiac autonomic nerves. Alterations of QT-RR relation after some respiratory manoeuvres 

are able to increase percentage of prolonged QT intervals over the upper limit (6). 

The latest investigations (7, 8) show that the QT-RR relation is not uniform but can frequently 

be very individual with intersubject differences. There are some conditions like carrying or 

lifting heavy objects, constipation, severe coughing spells, nausea, and vomiting, that increase 

intrathoracic pressure like Valsalva manoeuvre (VM) and they may prolong the QT interval duration 

or increase QT dispersion. VM consists of 4 phases of cardiovascular changes (9). 

The purpose of this study was to investigate if abrupt changes in autonomic tone are able to 

modulate the relation between durations of QT interval and cycle length during VM in young 

healthy women. 

Address for correspondence: 

Assoc. Prof. ·tefan Kujaník, M.D., Ph.D., , Department of Physiology, Medical Faculty of P. J. ·afárik University, 

Trieda SNP 1, 040 66 Ko‰ice, Slovak Republic 

Phone: ++421 55 6423 763, Fax: ++421 55 6420 253 

e-mail: kujanik@central.medic.upjs.sk

4 


METHODS 

Twenty-eight young healthy non-obese non-smoking female volunteers (18-24 years) 

were studied. They had a negative result of preventive medical examination, their history was 

without any serious disease, their cardiac and pulmonary auscultation findings or blood pressure 

were within a normal range of values. The non-obese women were chosen because a more 

expressive obesity decreases the parasympathetic activity and increases the sympathetic predominance. 

They were also not suffering from anorexia nervosa. Obesity prolongs QTc interval and 

increases the catecholamine level (10) and anorexia nervosa increases the QT dispersion (11), i.e. 

both act proarhythmically. Our volunteers refrained from alcohol for 24 hours and coffee, tea, 

cola drinks or a heavy meal for 6 hours before examination. Vigorous physical activity was avoided 

on the day of the study. 

Our young volunteers were examined during resting normal ventilation (RNV = control values) 

and during total VM in the recumbent position with tilt head up by 60 grades. The basal recordings 

(RNV = control phase) were obtained after at least 5 minutes of rest, breathing frequency 

was controlled at 12-14 cycles/minute. The VM was performed by using a mouthpiece connected 

to manometer, expiration pressure of 40 mmHg (5.33 kPa) was sustained for 20 seconds. 

The electrocardiogram in Frank leads (X, Y, Z) and standard limb leads (I, II, III) was recorded 

by the device Chiracard 600T (Chirana) continuously during RNV and VM at a paper speed 

of 100 mm/s, calibration 1 mV = 1.5 cm, the T-P interval was defined as the isoelectric baseline. 

The QT interval was measured from the earliest onset of the QRS complex in any lead to the 

latest end of the T wave in any lead, defined as the return to baseline. The measured QT and 

preceding RR intervals were always measured during the total VM manually. It demonstrated 

(12) an excellent agreement between manual and automated measurements. Division into 4 phases 

of VM was made according to changes of heart rate. The values obtained from five consecutive 

beats were averaged for every phase of VM. If U wave was present, the end of the T wave was 

measured according to the principles described by Lepeschkin and Surawicz (13). The values of 

QTc over 440 ms were considered pathologic (14). The QT dispersion was not measured. 

The measurements were performed in the daytime (9:00 to 17:00), i.e. during period of higher 

QT variability (15). QTc interval was calculated according to the Bazett`s formula (1) QTc = 

QT/square root of the preceding RR interval. Correlation between measured QT and its corresponding 

RR interval was tested by the value of simple linear correlation coefficient „r“. The investigation 

conforms to the principles outlined in the Declaration of Helsinki. The group „Total VM“ 

was created as the average result from all four phases of VM. 

The statistical processing was performed by the program Microsoft Excel in Microsoft Office 

97. The numerical data were expressed as arithmetic mean ± one SD, the statistical significance 

of differences was tested by the paired t-test compared to RNV. 

RESULTS 

a) Normal ventilation at rest (RNV = control): 

During RNV any extrasystoles did not occur. The RR, heart rate (HR), measured QT, and QTc 

intervals were within the normal electrocardiographic values (Table 1). The average HR ranged 

from 48 to 90 per min, average RR from 667 to 1250 ms, average QT from 322 to 452 ms, averaged 

QTc from 381 to 433 ms in this phase. The average QTc intervals were normal (under 440 

ms) in all persons. Relation between QT and RR intervals was individual, the same duration of 

QT occurred with different RR or heart rates (HR). The durations of studied parametres at rest 

were considered 100% and compared with alterations during VM. Correlation between measured 

QT and RR intervals was significant (r = 0.8948; p < 0.01) during RNV. 

b) The first phase of VM: 

No extrasystoles occurred in the first phase of VM. Duration of QT and RR intervals shortened 

(because of tachycardia) but not proportionally, QT interval was more stable. This phase was


the most variable for RR intervals since the highest value of SD occurred here. Compared to RNV 

the HR, RR, QT and QTc intervals were significantly different (p < 0.001; Table 1), their average 

values were the smallest compared to RNV. The average HR ranged from 49 to 139 per min, average 

RR from 431 to 1220 ms, average QT from 307 to 450 ms, averaged QTc from 372 to 494 

ms in this phase. The average QTc interval was over 440 ms in 12 volunteers (42.86%). Correlation 

between the measured QT and RR intervals was slightly less significant (r = 0.8196; p < 

0.01) compared to RNV. 

c) The second phase of VM: 

Extrasystoles did not occur here. This phase was the most variable (the highest value of SD) 

for QT interval. The RR, QT, QTc intervals and HR were significantly different (p < 0.001) compared 

to RNV. The average HR ranged from 66 to 160 per min, average RR from 375 to 906 ms, 

average QT from 293 to 450 ms, averaged QTc from 380 to 516 ms in this phase. Normal duration 

of the average QTc intervals (under 440 ms) was in 8 volunteers (28.57%) only. Correlation 

between the measured QT and RR intervals was significant (r = 0.8169; p < 0.01). 

d) The third phase of VM: 

Extrasystoles did not occur here. Compared to RNV the HR, RR, QT and QTc intervals were 

significantly different (p < 0.001), the maximum differences compared to RNV from all VM phases 

occurred here. The average HR ranged from 69 to 172 per min, average RR from 349 to 867 

ms, average QT from 288 to 422 ms, averaged QTc from 405 to 519 ms in this phase. Duration 

of average QTc intervals was normal (under 440 ms) in 5 persons (17.86%) only. Correlation between 

the measured QT and RR intervals was significant (r = 0.8381; p < 0.01). 

e) The fourth phase of VM: 

Extrasystoles did not occur here. Heart rate was the most variable in this phase (the highest 

value of SD), fast alterations of RR intervals from one cardiac cycle to other one accompanied 

with small QT alterations were present here (Figure 1). Compared to RNV the HR, RR, QT and 

QTc intervals were significantly different (p < 0.001). The average HR ranged from 71 to 196 per 

min, average RR from 306 to 850 ms, average QT from 303 to 430 ms, averaged QTc from 400 

to 547 ms in this phase. Duration of average QTc intervals was under 440 ms in 11 persons 

(39.29%) only. Correlation between the measured QT and RR intervals was significant (r = 

0.7506; p < 0.01). 

Table 1. QT-RR correlation during Valsalva manoeuvre (arithmetic mean ± one SD) in young healthy women (n = 28). 

Statistical significance compared to the resting values. HR – heart rate, QT – measured QT interval, QTc – corrected QT 

interval according to the Bazett formula, RNV – resting normal ventilation, „r“ – coefficient of simple linear correlation, 

VM – Valsalva manoeuvre. 

Period HR [beat/min] RR [ms] QT [ms] QTc [ms] „r“ QT to RR 

RNV (control) 77.04 ± 9.07 791.3 ± 116.0 364.4 ± 26.5 410.7 ± 13.1 0.8948 

p< 0.01 

VM – Phase 1 98.1 ± 17.4 635.6 ± 144.2 346.1 ± 29.1 437.8 ± 25.6 0.8196 

p< 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.01 

VM – Phase 2 108.8 ± 23.8 579.3 ± 135.7 342.5 ± 31.8 455.1 ± 32.0 0.8169 

p< 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.01 

VM – Phase 3 118.0 ± 25.6 534.2 ± 127.1 335.0 ± 29.2 463.5 ± 30.9 0.8381 

p< 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.01 

VM – Phase 4 112.3 ± 28.2 563.5 ± 126.2 337.7 ± 28.6 454.2 ± 37.3 0.7506 

p< 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.01 

VM – 4 phases 112.6 ± 24.2 557.9 ± 125.8 340.3 ± 27.8 460.7 ± 32.7 0.7855 

together p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001

6 


Table 2. The average alterations of the heart rate (HR), RR, QT and QTc intervals compared to the resting normal ventilation 

(RNV = 100%) during all phases of Valsalva manoeuvre (VM) in the young healthy women (n = 28). * – the minimum 

average alteration, ** – the maximum average alteration. 

Time period Mean HR (%) Mean RR (%) Mean QT (%) Mean QTc (%) 

RNV (control) 100 100 100 100 

1 st phase of VM 127.27* 80.33* 94.97* 106.61* 

2 nd phase of VM 141.23 73.22 94.0 110.81 

3 rd phase of VM 153.17** 67.51** 91.92** 112.86** 

4 th phase of VM 145.77 71.66 92.69 110.60 

Total VM 146.16 70.50 93.40 112.19 

f) The total VM (all four phases together): 

During the total VM no extrasystoles occurred. All measured parameters were significantly 

different (p < 0.001) from RNV. The average HR ranged from 67 to 171 per min, average RR from 

351 to 896 ms, average QT from 302 to 438 ms, averaged QTc from 394 to 535 ms. Duration of 

average QTc interval was normal (under 440 ms) in 5 women (17.86%) only. Correlation between 

measured QT and RR intervals was significant (r = 0.7855; p < 0.01). 

The results of all studied parameters are shown in the tables and one figure. Compared to 

RNV the heart rate, RR interval, QT and QTc values altered significantly during all phases of VM 

(Table 1), the largest relative alterations in % were present in HR, the smallest ones in measured 

QT (Table 2). The RR intervals sometimes altered very markedly from one cardiac cycle to the 

other one while the QT interval altered almost not distinctly. Sometimes the QT and RR intervals 

were in the opposite relation, i.e. QT was stable but RR altered very markedly or one parameter 

was shortening and the second one prolonging and vice versa (Figure 1). The correlation between 

QT and RR intervals was significant (p < 0.01) in all phases, it was the highest (r = 0.8948) 

at rest and slightly lower during other phases (r = 0.8196, 0.8169, 0.8381, 0.7506 or 0.7855, 

Table 1). The independence between the duration of QT and RR intervals was the most distinct 

in the phase 4 (Figure 1). There was a special relationship time to time – RR was shortening but 

QT prolonging in the same time. 

DISCUSSION 

The ventricular repolarization is inhomogenous, its differences exist between the left and 

right ventricle, between the epicardium, mid-myocardium (M cells), and endocardium, and between 

the cardiac base and apex. These differences (increased or decreased) are influenced by 

various physiologic, pharmacologic, and pathologic interventions (16), such as autonomic tone, 

hypoxia, ischaemia, cardiac hypertrophy, temperature, drugs or ionic imbalance. It was demonstrated 

that the QT-RR relationship pattern varied significantly already among healthy individuals 

but their intraindividual stability was observed as well (7). This finding can be proved by 

our measurements. Statistical significance of the QT interval differences is also dependent on the 

mode of QT expressing (measured QT, corrected QT or QT together with its heart rate – 17). Ratecorrection 

formulae are proposed to allow interindividual comparisons at different HRs. 

We studied the female gender only which is considered to be a risk factor for ventricular arrhythmias. 

Clinical and experimental observations suggest the existence of true differences in 

electrophysiologic properties between the sexes. Estrogen has an impact on the electrophysiological 

properties of the heart. The progestin-oestrogen replacement therapy significantly reduces 

ventricular QT-dispersion compared to the control group, while only oestrogen replacement the-


Figure 1. Arelatively very independent beat-to-beat interrelation between QT and RR intervals during Valsalva manoeuvre 

in one of our young female volunteers. 

rapy significantly prolongs QTc - intervals without affecting QT dispersion (18). At physiological 

resting heart rates, the spatial ST-T vector voltage time trajectory is steeper in men than in 

women (19). Since the QT and RR intervals alter with heart rate, many formulae for QT correction 

(removing the rate dependence) were introduced within 80 years. The Bazett’s formula for 

QTc calculation merely diminishes but does not remove the rate dependence (Table 2). 

There is a different autonomic innervation of the heart. The vagus nerves supply predominantly 

the atria and conductive system of the heart and influence mostly the cardiac HR or RR 

intervals and conduction velocity in the atria. The chronotropic parasympathetic influence is realized 

mainly through the right vagus nerve (20) acting predominantly on the sinus node. A vagal 

nerve stimulation exerts only minimum effects on ventricular functions. In ventricles the sympathetic 

nerves influence the QT interval duration, excitability, and contractility. A recent study 

(21) shows heterogeneity of sympathetic innervation in various kinds of pathological conditions 

in normal human heart – the inferioposterior region shows distinctly less sympathetic innervation 

than the anterior region. 

The effect of autonomic nerves on the heart and QT interval is complex but the cardiac autonomic 

blockade in ganglia prolongs QTc interval (22). Parasympathetic postganglionic acetylcholine 

is removed very rapidly from the muscarinic receptors by acetylcholin-esterase and alterations 

of RR interval are substantially different already from beat to beat (fast cardiac control). 

Sympathetic postganglionic noradrenalin is metabolized longer (slow cardiac control), most of its 

amount is reuptaken and QT duration is not substantially altered within several cardiac cycles 

(23). However, the RR interval duration is not a result of quantity of sympathetic or parasympathetic 

activity. There are no physiological evidences that the levels of sympathetic and vagal 

nerve fluctuations are balanced (24).

8 


In contrast to cardiac depolarization, the repolarization phase cannot be described in terms 

of wavefront propagation. The QT duration such as RR interval may also be influenced by the 

day-time (during sleep it is longer) or by short-term variations of the T wave form. The QT dispersion 

measurement may be due to measurement errors (25) and low amplitude potentials are 

undetectable in some leads. It cannot be assumed that autonomic influences on the atrial pacemaker 

structures and on the ventricular myocardium are acting in parallel and RR interval can 

not give us information on the state of the autonomic regulation of the cardiac ventricles. The 

cardiac autonomic fibres are divided according to their function. Already in the first half of twentieth 

century the different branches of the cardiac plexus were named according to their main 

function (acceleratory, slowing or strengthening nerves). 

For mathematical expression of the relation between QT and RR intervals many authors have 

proposed linear or non-linear regression equations since 1920 (1), however, all of them have 

some limitations. It seems that the search for a universally applicable QT/RR regression model 

that would provide the best fit in all circumstances is most likely fruitless (26). Within a wide 

range of RR intervals the QT duration in reflex cardiovascular reactions is altering only very 

slightly (3, 4, 5). Our finding proves the opinion of these authors. When heart beats are selected 

for a steady rhythm during the preceding minute, QT and RR intervals fit a linear relationship 

during the day and night periods, but not during the 24-hour period. In contrast, in the absence 

of beat selection, data fit a more complex curvilinear relationship irrespective of the period 

(12). The autonomic conditions may probably directly affect the ventricular myocardium of healthy 

subjects, causing differences in QT that are independent of HR (27). QT rate dependence is 

larger during the day in both genders in healthy subjects (28). Women show stronger QT rate 

dependence and the circadian modulation decreases with increasing age. 

It seems that QT interval dispersion measured from the body surface is not a reliable index 

of repolarization dispersion in ventricular myocardium (29) and QT dispersion from body surface 

ECG does not reflect the spatial dispersion of ventricular repolarization (30, 31). Dispersion 

of the QT interval and other ECG variables of dispersion of ventricular repolarization are independent 

on heart rate. Therefore, it is not necessary to rate-correct those measurements of dispersion 

(32). However, QT dispersion has a dynamic behaviour with significant beat-to-beat fluctuations 

even in normal subjects (33). 

REFERENCES 

1. Bazett HC. An analysis of the time relations of electrocardiograms. Heart 1920; 7: 353-370. 

2. Malik M. The imprecision in heart rate correction may lead to artificial observations of drug induced QT interval 

changes. Pacing Clin Electrophysiol 2002; 25 (2): 209-216. 

3. Anderson RC. Q-T interval in sinus arrhythmia. J Electrocardiol 1981; 14 (4): 407-408. 

4. Davidowski TA, Wolf S. The QT interval during reflex cardiovascular adaptation. Circulation 1984; 69 (1): 22-25. 

5. Kujaník ·. QT/QS 2 

pri zmenách pºúcnej ventilácie u zdrav˘ch ºudí. [QT/QS 2 

during pulmonary ventilation alterations 

in healthy humans]. Noninvas Cardiol 1994; 3 (3): 149-152. 

6. Kujaník ·, Valachová A, Kubáãek ·, Mikuleck˘ M. Dependence of QT interval on the heart rate during alterations of 

pulmonary ventilation in young healthy subjects. Physiol Res 1993; 42 (6): 383-389. 

7. Batchvarov VN, Ghuran A, Smetana P, Hnatkova K, Harries M, Dilaveris P, Camm AJ, Malik M. QT-RR relationship 

in healthy subjects exhibits substantial intersubject variability and high intrasubject stability. Am J Physiol - Heart 

Circ Physiol 2002; 282 (6): H2356-H2363. 

8. Malik M, Farbom P, Batchvarov V, Hnatkova K, Camm AJ. Relation between QT and RR intervals is highly individual 

among healthy subjects: implications for heart rate correction of the QT interval. Heart 2002; 87 (3): 220-228. 

9. Nishimura RA, Tajik AJ. The Valsalva maneuver and response revisited. Mayo Clin Proc 1986; 61 (3): 211-217. 

10. Corbi GM, Carbone S, Ziccardi P, Giugliano G, Marfella R, Nappo F, Paolisso G, Esposito K, Giugliano D. FFAs and 

QT intervals in obese women with visceral adiposity: Effects of sustained weight loss over 1 year. J Clin Endocrinol 

Metab 2002; 87 (5): 2080-2083. 

11. Galetta F, Franzoni F, Cupisti A, Belliti D, Prattichizzo F, Rolla M. QT interval dispersion in young women with anorexia 

nervosa. J Pediatrics 2002; 140 (4): 456-460. 

12. Lande G, Funckbrentano C, Ghadanfar M, Escande D. Steady-state versus non-steady-state QT-RR relationships in 

24-hour Holter recordings. Pacing Clin Electrophysiol 2000; 23 (3): 293-302. 

13. Lepeschkin E, Surawicz B. The measurement of the QT interval of the electrocardiogram. Circulation 1952; 6: 378- 

388.


14. Moss AJ, Schwartz PJ. Sudden death and the idiopathic long QT syndrome. Am J Med 1979; 66 (1): 6-7. 

15. Kostis WJ, Belina JC. Differences in beat-to-beat variability of the QT interval between day and night. Angiology 

2000; 51 (11): 905-911. 

16. Gettes LS. The T wave: A window on ventricular repolarization? J Cardiovasc Electrophysiol 2001; 12 (11): 1326- 

1328. 

17. Kujaník ·, Valachová A, Mikuleck˘ M, Murín M. Dependence of statistical significance of QT interval differences on 

the mode of QT expressing. Folia Fac Med Univ ·afarik Cassov 1990; 47: 83-88. 

18. Haseroth K, Seyffart K, Wehling M, Christ M. Effects of progestin-estrogen replacement therapy on QT-dispersion in 

postmenopausal women. Int J Cardiol 2000; 75 (2-3): 161-165. 

19. Lehmann MH, Yang H. Sexual dimorphism in the electrocardiographic dynamics of human ventricular repolarization 

– characterization in true time domain. Circulation 2001; 104 (1): 32-38. 

20. Rothberger C. Allgemeine Physiologie des Herzens. In: Handb norm u pathol Physiol, Bd 7/1, Berlin 1926: 523-662. 

21. Momose M, Tyndalehines L, Bengel FM, Schwaiger M. How heterogeneous is the cardiac autonomic innervation? 

Basic Res Cardiol 2001; 96 (6): 539-546. 

22. Diedrich A, Jordan J, Shannon JR, Robertson D, Biaggioni I. Modulation of QT interval during autonomic nervous 

system blockade in humans. Circulation 2002; 106 (17): 2238-2243. 

23. Van Ravenswaaij-Arts CMA, Kollée LAA, Hopman JCW, Stoelinga GB, van Geijn HP. Heart rate variability. Ann Intern 

Med 1993; 118 (6): 436-446. 

24. Ruttkay-Nedeck˘ I. The effect of the autonomic nervous system on the heart. Electrocardiographic evaluation: Problems 

and concerns. Cardiol (Bratislava) 2001; 10 (1): 42-48. 

25. Kors JA, van Herpen G. Measurement error as a source of QT dispersion: a computerized analysis. Heart 1998; 80 

(5): 453-458. 

26. Hnatkova K, Malik M. „Optimum’’ formulae for heart rate correction of the QT interval. Pacing Clin Electrophysiol 

1999; 22 (11): 1683-1687. 

27. Magnano AR, Holleran S, Ramakrishnan R, Reiffel JA, Bloomfield DM. Autonomic nervous system influences on QT 

interval in normal subjects. J Am Coll Cardiol 2002; 39 (11): 1820-1826. 

28. Extramiana F, Maison-Blanche P, Badilini F, Pinoteau J, Deseo T, Cournel P. Circadian modulation of QT rate dependence 

in healthy volunteers: gender and age differences. J Electrocardiol 1999; 32 (1): 33-43. 

29. Lux RL, Fuller MS, Macleod RS, Ershler PR, Green LS, Taccardi B. QT interval dispersion. Dispersion of ventricular 

repolarization or dispersion of QT interval? J Electrocardiol 1998; 30(Suppl): 176-180. 

30. Wang LX. QT dispersion from body surface ECG does not reflect the spatial dispersion of ventricular repolarization 

in sheep. Pacing Clin Electrophysiol 2000; 23 (3): 359-364. 

31. Malik M, Acar B, Gang Y, Yap YG, Hnatkova K, Camm AJ. QT dispersion does not represent electrocardiographic 

interlead heterogeneity of ventricular repolarization. J Cardiovasc Electrophysiol 2000; 11 (8): 835-843. 

32. Zabel M, Franz MR, Klingenheben T, Mansion B, Schultheiss HP, Hohnloser SH. Rate-dependence of QT dispersion 

and the QT interval: Comparison of atrial pacing and exercise testing. J Am Coll Cardiol 2000; 36 (5): 1654-1658. 

33. Figueredo EJG, Ohnishi Y, Yoshida A, Yokoyama M. Usefulness of beat-to-beat QT dispersion fluctuation for identifying 

patients with coronary heart disease at risk for ventricular arrhythmias. Am J Cardiol 2001; 88 (11): 1235- 

1239. 

Received: May, 25, 2003 

Accepted: June, 19, 2003

10 


CHEMICAL CONTROL OF BREATHING IN ANAESTHETIZED 

RABBITS DURING HYPERTHERMIA AND ITS RECOVERY BY BODY 

SURFACE COOLING 

IVAN ÎILA, ANDREA BROZMANOVÁ, KAMIL JAVORKA, MICHAL JAVORKA, 

ANDREA âALKOVSKÁ, MÁRIA PETRÁ·KOVÁ 

Department of Physiology, Comenius University, Jessenius Faculty of Medicine, Faculty Hospital Martin, Slovak Republic 

Abstract 

The contribution of chemical control mechanisms in the development of respiratory changes during hyperthermia 

and its recovery by body surface cooling was studied in 14 adult rabbits. 

Hypercapnic (HCVR) and hypoxic ventilatory responses (HVR) were estimated during body surface heating and cooling. 

HCVR: CO 2 

-sensitivity in normothermia was 115 ± 22 ml.min -1 .kPa -1 (mean ± SEM). During overheating the sensitivity 

was significantly increased – at 42 o C it was 162 ± 20 ml.min -1 .kPa -1 . Recovery of body temperature (BT) was not 

accompanied with significant change of CO 2 

-sensitivity. HVR: gradual decrease of FiO 2 

during overheating caused rise 

of ventilation lesser than in normothermia. During cooling, there was similar change of ventilation during episodes of 

hypoxia. As temperature recovered to the initial value, ventilation did not significantly change with decrease of FiO 2 

. 

Minute ventilation significantly increased only compared to V E 

in normoxia. 

The results indicate that during hyperthermia HCVR was augmented and HVR was attenuated. Attenuation of HVR 

persists during recovery of BT, while HCVR did not significantly change. 

Key words: chemical control of breathing, chemoreflex sensitivity, hyperthermia, hypoxia, hypercapnia 

INTRODUCTION 

The changes of body temperature (BT) are accompanied with marked alterations of respiratory 

parameters. It is supposed that some of these changes are evoked by alterations in the 

peripheral temperature. The other changes are modified by shifts in the central BT, eventually 

by other mechanisms (1). However, previous reports considerably interpret various evidence of 

contribution of chemoreflexes in the origin of respiratory instability in hyperthermia. 

The effects of respiratory responses to a lowering of BT are often complicated by the conditions 

of body cooling (1). Recovery of BT to normothermia is accompanied by a gradual resetting 

of respiratory parameters near to the initial values and acceleration of recovery (e.g. by cooling) 

elicits further cardiorespiratory changes (2). These responses are rather more complex. Elevated 

thermogenesis requires increased oxygen uptake which is as a consequence often associated 

with increased ventilation (V E 

). Galland (1991) reports that recovery of BT from hyperthermia to 

the initial value is considered to be a phase of impaired control of breathing. 

The present study was undertaken to obtain information on the contribution of chemical control 

mechanisms to the development of respiratory changes in experimental hyperthermia and 

its physical treatment in rabbits. 

METHODS 

The experiments were carried out on 14 adult rabbits, body weight (b.w.) 2.6 ± 0.1 kg (mean 

± SEM). The animals were anaesthetized with intramuscular ketamine (Spofa, Czech Republic) 

at a dose of 25 mg/kg b.w. and xylazine (Spofa) at a dose of 5 mg/kg b.w. followed by continu- 


Ivan Îila, M.D., Department of Physiology, Comenius University, Jessenius Faculty of Medicine, Faculty Hospital, 

Malá Hora 4, 037 54 Martin, Slovak Republic 

Phone: ++421 43 4131 426, Fax: ++421 43 4222 260 

e-mail: zila@jfmed.uniba.sk


ous intravenous infusion of ketamine at a dose of 20 mg/kg/hour. The animals were tracheotomized 

and breathed spontaneously room air through a tracheal cannula. The tidal volume (V T 

) 

was recorded by the Fleisch head of a pneumotachograph (ÚMMT SAV, Bratislava) connected to 

the tracheal cannula. End-tidal CO 2 

(ETCO 2 

) was continuously recorded using mainstream sensor 

of a capnograph (Capnogard, Novametrix, USA) connected to the head of pneumotachograph. 

The frequency of breathing (f) was calculated from the tidal volume recording. Blood pressure 

in the femoral artery was recorded with the electromanometer LDP 102 (Tesla, Czech Republic). 

Blood samples were taken from the femoral artery for the blood gases (p a 

O 2 

, p a 

CO 2 

) and pH a 

analysis 

using a blood gas analyzer (Radiometer, Denmark) and corrected for actual BT. The catheter 

in the femoral vein was used for a continuous administration of the anaesthetic by the injection 

pump IPA 2050 (COMPACT Co., Czech Republic). Rectal temperature was measured with 

mercury thermometer at a depth of 6-7 cm. 

The experiment was divided to two phases. In the first one, initial body temperature of the 

animal (T 1 

) was gradually elevated by surface heating to 42.0 o C using a heating pad and radiant 

heat from an infrared lamp. Subsequently, body surface cooling by a cooling pad and wet cold 

wraps were used for recovery of BT to the initial value. 

Hypercapnic and hypoxic ventilatory response was measured during body surface heating at 

39.5-40.5 o C (T 2 

) and 42.0 o C (T 3 

) as well as during cooling at 40.5-39.5 o C (T 4 

) and when BT recovered 

to the initial value (T 5 

= T 1 

). 

Hypercapnic ventilatory response (HCVR). The animals breathed from a bag with gas mixture 

of 40% O 2 

balanced with N 2 

. For continuous rise of end-tidal CO 2 

(ETCO 2 

), CO 2 

was added to the 

inspiratory gas. Gradual rising of CO 2 

-tension was performed in 2 minutes time period and endtidal 

CO 2 

was monitored in 10 second intervals. Hence, 12 values of ETCO 2 

were ploted against 

corresponding data from pneumotachograph. CO 2 

sensitivity was estimated as the slope of ventilation 

– ETCO 2 

curves. Blood samples were taken between 50-60 and 110-120 second of CO 2 

run. 

Hypoxic ventilatory response (HVR). After 3 minutes of resting period (animals breathed room 

air), second phase of protocol started. The animals breathed from four bags containing gas mixture 

of 11%, 9%, 7% and 5% O 2 

, balanced with N 2 

(H 11 

, H 9 

, H 7 

, H 5 

). Each mixture was inhaled 

for 2 minutes, isocapnia was performed by manually controlled addition of CO 2 

to inspiratory 

gas. Between each hypoxic mixture there was 30 second period of room air breathing. Blood 

samples were taken at the same time intervals as for the CO 2 

run. The HVR was estimated as 

the change of ventilation (V E 

) regarding ventilation in normoxia and ventilation during episodes 

of hypoxia. V E 

was assessed at the end of the first and the second minute of hypoxic runs. 

The rabbits were killed by overdosing with the anaesthetic drug et the end of the experiment. 

Experiments were done according to Helsinki Declaration of 1975, revised in 1983. 

Statistical analysis: Statistical analysis was performed using a Wilcoxon test to evaluate within-group 

changes. The results are expressed as means ± SEM. Differences were considered significant 

when P < 0.05. 

RESULTS 

Hypercapnic ventilatory response – central chemoreflex sensitivity 

CO 2 

sensitivity in normothermia was 115 ± 22 ml.min -1 .kPa -1 . During overheating, at T 2 

no 

significant change was found (154 ± 19 ml.min -1 .kPa -1 ). At 42 o C (T 3 

) sensitivity was significantly 

increased (162 ± 20 ml.min -1 .kPa -1 vs. normothermia, p < 0.05). During recovery of BT the sensitivity 

of the central chemoreflex did not change significantly (T 4 

: 159 ( 22 ml. min -1 .kPa -1 , T 5 

: 

158 ± 24 ml.min -1 .kPa -1 ; Figure 1). 

Hypoxic ventilatory response 

While hypoxic stimulation in normothermia evoked a gradual rise of ventilation (V E 

), change 

of BT during the hypoxic run led to reduction of differences in V E 

. With increased intensity of

12 


Figure 1. CO 2 

-sensitivity during overheating (T 2 

= 

39.5-40.5 o C, T 3 

= 42 o C) and cooling (T 4 

= 40.5- 

39.5 o C, T 5 

= initial value) compared to normothermia 

(T 1 

). 

Figure 2. Hypoxic ventilatory response. Change of ventilation 

during normoxia (FiO 2 

= 0.21) and episodes of hypoxia at different 

degrees of body temperature. 

hypoxia V E 

rose slowly with change of BT from T 1 

to T 5 

(Figure 2). . 

Breathing a gas mixture containing 

5% O 2 

(H 5 

) elicited a significant rise of V E 

only in comparison with V E 

in normothermia 

(Table 1). With the rise of intensity of hypoxia from 11% O 2 

(H 11 

) to 5% O 2 

(H 5 

) changes of tidal 

volume (V T 

) at T 1 

-T 5 

were more accentuated. During overheating V T 

decreased more rapidly at 

lower concentration of O 2 

in inspired gas and similarly rose more rapidly with recovery of BT to 

the initial value. While V T 

at H 11 

did not change during overheating, at H 7 

and H 5 

there was a significant 

decrease between V T 

at T 3 

and T 1 

and T 2 

-T 3 

. Frequency of breathing (f) during hypoxic 

run changed with rise and decrease of BT by the same manner as frequency in normoxia. The 

recovery phase was accompanied by a significant decrease of f at all hypoxic mixtures. 

p a 

O 2 

at the beginning of the experiments was 10.9 ± 0.83 kPa, during overheating decreasedat 

T 3 

it was 8.1 ± 0.29 kPa (p < 0.05). In the course of recovery of BT to the initial value significant 

increase was found. Episodes of hypoxia were accompanied with gradual decrease of p a 

O 2 

. 

In three cases, decrease of FiO 2 

was not followed by significant decrease of p a 

O 2 

(Table 2). 

DISCUSSION 

Hypercapnic ventilatory response (HCVR) 

Our results show that a rise in the BT led to the increase of HCVR. CO 2 

-sensitivity at 42 o C 

significantly increased compared to value at initial BT. This finding is in accordance to studies 

performed both in humans and in animals (4, 5, 6). However, some investigators have found no 

change of central chemoreflex sensitivity (7). In some experiments a decrease of HCVR in a warm 

environment was observed (8). There are several possible reasons for contradictory observations, 

but the different methods of investigation seem to be the most considerable reason for the discrepancy 

of the results. 

An increase of CO 2 

-sensitivity (between T 1 

and T 3 

) could be explained by at least two mechanisms. 

Firstly, increased metabolic drive can be responsible for augmented HCVR. Secondly, 

there is an assumed interaction between thermal and central chemoreceptor drives to breathe 

(6). This relation is multiplicative rather than additive and authors suggest that there is also an 

additive ventilatory drive component due to thermal stimuli independent of carbon dioxide (6). 

Recovery of BT from hyperthermia to initial value is considered to be a phase of impaired control 

of breathing (3). In our study there was also a tendency to decrease CO 2 

-sensitivity in this 

period, however, the differences were not statistically significant. Some experiments show that 

in anaesthetized animals, mild hypothermia does not affect the response to CO 2 

, however, the 

use of deep hypothermia does provide some evidence of reduced CO 2 

-sensitivity (9).


Table 1. Hypoxic ventilatory response. Minute ventilation (ml/min) in normothermia (T 1 

), during overheating (T 2 

= 39.5- 

40.5 o C, T 3 

= 42 o C) and during body temperature recovery (T 4 

= 39.5-40.5 o C, T 5 

= initial value). 

normoxia H 11 

H 9 

H 7 

H 5 

T 1 

1051.4 ± 120.6 1399.6* ± 174.1 1542.3* ± 167.3 1760.6* ± 196.1 1872.4* ± 184.6 

T 2 

1502.8† ± 158.3 1979.5*† ± 240.3 2116.1*† ± 281.3 2187.3*† ± 278.8 2277.9† ± 293.9 

T 3 

1866.5† ± 223.0 2316.9*† ± 212.8 2369.4† ± 238.3 2364.4 ± 260.2 2329.9 ± 248.9 

T 4 

1822.0 ± 162.3 2254.7* ± 241.1 2329.2 ± 299.8 2448.4 ± 276.7 2486.5 ± 268.5 

T 5 

1996.4 ± 165.9 2301.3* ± 193.4 2319.1 ± 199.7 2298.9 ± 231.4 2363.8 ± 252.8 

Values are shown as means ± SEM. 

* Value is significantly greater than previous value in row, P < 0.05 

† Value is significantly greater than previous value in column, P < 0.05 

Table 2. Hypoxic ventilatory response. paO 2 

(kPa) in normothermia, during overheating (T 2 

= 39.5-40.5 o C, T 3 

= 42 o C) 

and during body temperature recovery (T 4 

= 40.5-39.5 o C, T 5 

= initial value). 

normoxia H 11 

H 9 

H 7 

H 5 

normothermia 10.9 ± 0.8 7.3 ◊ ± 0.5 6.6 ◊ ± 0.3 6.0 ◊ ± 0.2 5.4 ◊ ± 0.2 

T 2 

9.4 ± 0.2 5.9 ◊ ± 0.2 5.6 ± 0.2 5.1 ◊ ± 0.2 4.5 ◊ ± 0.2 

T 3 

8.1 ± 0.3 5.5 ◊ ± 0.3 4.8 ◊ ± 0.2 4.4 ◊ ± 0.2 3.9 ◊ ± 0.2 

T 4 

10.0 † ± 0.4 7.0 ◊† ± 0.3 6.4 ◊† ± 0.3 6.0 † ± 0.4 5.4 ◊† ± 0.3 

T 5 

13.2 † ± 0.7 8.9 ◊† ± 0.5 8.6 † ± 0.7 7.6 ◊† ± 0.5 6.6 ◊† ± 0.4 

Values are shown as means ± SEM 

*Value is significantly greater than previous value in row, p < 0.05 

◊ Value is significantly lesser than previous value in row, p < 0.05 

† 

Value is significantly greater than previous value in column, p < 0.05 

 

Value is significantly lesser than previous value in column, p < 0.05 

Hypoxic ventilatory response (HVR) 

In this study, the rise in BT was accompanied with attenuation of HVR. While at initial BT 

minute ventilation (V E 

) gradually increased with rise of intensity of hypoxia, at T 2 

increasing of 

V E 

was moderate. Hypoxic stimulation at T 3 

evoked the increase of V E 

only in comparison to the 

initial minute ventilation. 

We observed that, whereas changes of V E 

in normothermia were due to proportional change 

of tidal volume (V T 

) and frequency of breathing (f), during overheating (T 2 

) less marked change of 

V T 

was present. Frequency of breathing increased during episodes of hypoxia, but mostly in 

cases of mild hypoxia runs. At T 3 

, preferably in cases of severe hypoxia f decreased and therefore 

modest increase of V E 

was mainly due to rise of V T 

. 

Our findings are in accordance to study of Watanabe et al. (8), who showed that respiratory 

response mediated via peripheral chemoreceptors decreases in warmer environmental temperature 

in kittens. Authors assume the decrease in metabolism causing lower amplitude of oscillation 

in p a 

CO 2 

, and therefore decreasing intensity of respiratory response. Another possibility is 

an effect of the thermoreceptors on central nervous system. 

Recovery of BT to initial value was also accompanied with attenuation of HVR. Changes of V E

14 


were mostly due to increase of V T 

, frequency did not change, except 2 points when it decreased. 

At T 5 

ventilation did not change from mild to severe hypoxia. However, it increased in comparison 

to resting V E 

. During recovery of BT, decrease of fraction of inspired oxygen (FiO 2 

) slowed the 

increase of ventilation. Previous studies suggested that such a decrease could be due to hypometabolism 

caused by hypoxia. Decrease of metabolic drive to breathe might overcome hypoxic 

drive and lead to attenuation of HVR (10). In our experiments including 4 episodes of hypoxia, 

severe hypoxic runs could change the metabolism. Therefore we can not conclude that attenuation 

of HVR during recovery phase was only due to decrease in the gain of peripheral chemoreflex. 

Our results show that hyperthermia was accompanied with augmented HCVR and with attenuation 

of HVR. In spite of not significant decrease in CO 2 

-sensitivity during recovery of body 

temperature, attenuation of HVR persisted during cooling. Because of possible damage in 

mechanisms of control of breathing it would be worth to study the respiratory changes during 

physical treatment of hyperthermia. 

This work was supported by grant N. 1/6292/99 from Grant Agency VEGA (Slovak Republic) 

REFERENCES 

1. Cooper KE, Veale WL. Effects of temperature on breathing. Handbook of Physiology, The Respiratory system II, Chapter 

20, 1986. p. 691-699. 

2. Javorka K, Calkovska A, Petraskova M, Gecelovska V. Cardiorespiratory parameters and respiratory reflexes in rabbits 

during hyperthermia. Physiol Res 1996; 45: 439-447. 

3. Galland BC, Taylor BJ, Bolton DPG. Respiratory instability in the sleeping piglet following hyperthermia. Workshops 

Abstracts of the ESID Founding Congress, Rouen, 1991. p. 87. 

4. Widdicombe JG, Winning A. Effects of hypoxia, hypercapnia and changes in body temperature on the pattern of breathing 

in cats. Respir Physiol 1974; 21: 203-221. 

5. Maskrey M. Body temperature effects on hypoxic and hypercapnic responses in awake rats. Am J Physiol 1990; 259: 

492-498. 

6. Baker FJ,Goode CR, Duffin J. The effect of a rise in body temperature on the central-chemoreflex ventilatory response 

to carbon dioxide. Eur J Appl Physiol 1996; 72: 537-541. 

7. Vejby-Christensen H, Strange-Petersen E. Effect of body temperature and hypoxia on the ventilatory CO 2 

in man. 

Respir Physiol 1973; 10: 93-108. 

8. Watanabe T, Kumar P, Hanson MA. Effect of ambient temperature on respiratory chemoreflex in unanaesthetized kittens. 

Respir Physiol 1996; 106: 239-246. 

9. Cherniack NS, von Euler C, Homma I, Kao FF. Graded changes in central chemoreceptor input by local temperature 

changes on the ventral surface of medulla. J Physiol 1979; 287: 191-211. 

10. Mortola JP, Matsuoka T. Interaction between CO 2 

production and ventilation in the hypoxic kitten. J Appl Physiol 

1993; 74: 905-910. 

Received: February, 25, 2003 

Accepted: May, 25, 2003


NADPH: CYTOCHROME P450 REDUCTASES OF VARIOUS SPECIES CAN 

BE USED IN SYSTEMS RECONSTITUTING DRUG-METABOLIZING 

CYP2E1 ACTIVITY 

MARIE BELEJOVÁ, EVA ANZENBACHEROVÁ*, ROMAN ZUBER, PAVEL ANZENBACHER 

Institute of Pharmacology and *Institute of Medical Chemistry and Biochemistry, Faculty of Medicine, 

Palacky University, Olomouc, Czech Republic 

Abstract 

Metabolism of drugs and other foreign substances is mostly mediated by cytochromes P450 (P450, abbreviated also 

CYP for a particular enzyme). To find which P450 is involved in metabolism of a drug, liver microsomal monooxygenase 

system of P450 is reconstituted with its components: selected P450 enzyme, cytochrome b 5 

, NADPH:P450 reductase and 

phospholipid. Used NADPH:P450 reductases were human recombinant and minipig or rat liver microsomal ones isolated 

by chromatographic separations. Chosen P450 enzyme was the CYP2E1 which is known to be of very similar primary 

structure among species; in this study, the minipig enzyme has been taken, as the minipigs seem to be suitable model 

animals for drug metabolism studies. Results obtained show that the reductase enzymes of rat and human origin can 

be used in reconstituted systems with a CYP2E1 as the activity of this enzyme varies in systems with reductases of different 

origin less than ten times. The results also indicate that the reductases from different species share a functional 

similarity, if not identity. 

Key words: cytochrome P450, CYP2E1, NADPH:cytochrome P450 reductase, pig 

INTRODUCTION 

NADPH:cytochrome P450 oxidoreductase (abbreviated P450 reductase, EC 1.6.2.4) was found 

as an electron transporting flavoprotein in pig livers (1), lately, it has been determined to be 

amember of mammalian microsomal monooxygenase system of cytochromes P450 (2,3). Cytochromes 

P450 (P450, abbreviated also CYP for a particular enzyme) are known to mediate metabolism 

of drugs and other foreign substances and to take part in many biosynthetic pathways in 

organism (4). To find which P450 is involved in metabolism of a drug, liver microsomal monooxygenase 

system of P450 should be reconstituted with its components, namely, with the selected 

P450 enzyme, with the NADPH:P450 reductase and phospholipid, in some cases also with 

the cytochrome b 5 

(5). 

Three-dimensional structure of the rat liver microsomal P450 reductase has been determined 

recently (6,7). The P450 reductase is composed of two flavin-binding domains (the FMN- and 

FAD-binding one), of the domain which mediates the binding of the enzyme to the microsomal 

membrane and of the binding sites of the P450 and of the NADPH. However, for the use of P450 

reductases of different origin (i.e. from different species) in reconstituted microsomal P450 systems 

it is important to know whether they are mutually interchangeable. Although there are 

indications in the literature (5) that this is true at least for some activities (mainly when rabbit 

liver microsomal P450 reductase is used), it should be however proven for every particular case 

to be sure that the reconstitution is possible. This was also the main aim of this study: To prove 

the possibility to reconstitute the prototypical CYP2E1 activity (chlorzoxazone 6-hydroxylation 

(8), the P450 reductases from minipig and rat liver microsomes as well as the human recombinant 

reductase enzyme were used. 


Marie Belejová, MD, Institute of Pharmacology, Faculty of Medicine, Palacky University, 

Hnûvotínská 3, 775 15 Olomouc, Czech Republic 

Phone: 00421 58 563 2556 

e-mail: belejovm@fnol.cz

16 


MATERIAL AND METHODS 

The P450 reductases of the rat and minipig origin were isolated by affinity chromatography 

from solubilized liver microsomal fraction by a procedure based on method of Yasukochi and 

Masters (9). The rat microsomal fraction was obtained from Dr. Stiborová (Faculty of Sciences, 

Charles University, Prague). The human recombinant P450 reductase was purchased from Panvera 

(Woburn, MA, USA). 

The CYP2E1 was prepared from microsomal fraction of minipig liver homogenate by a procedure 

based on combination of various chromatographic steps as outlined by Guengerich (10), for 

a description of this approach applied to minipig P450 enzymes see (11). Cytochrome b 5 

was isolated 

as a by-product during the same isolation. The other materials were as a rule obtained 

from Sigma Aldrich CZ (Prague). The chlorzoxazone 6-hydroxy derivative was a product of Ultrafine 

Chemicals (Salford, UK). 

The experiments, in which the CYP2E1 enzyme activity with different P450 reductases was 

followed using a reconstituted system, were done according to procedure described in (5). Incubation 

mixture (0.250 ml) consisted of the CYP2E1 (20 pmol), the respective reductase (60 pmol), 

cytochrome b 5 

(80 pmol) and phospholipid (dilauroylphosphatidylcholine, 4 µg) in 50 mM K/PO 4 

buffer, pH 7.4) together with the NADPH-generating system (50 µl of 5 mM NADP + , 50 µl of 50 

mM glucose-6-phosphate, 5 µl of glucose 6-phosphate dehydrogenase, 100 µl of 1.0 M K/PO 4 

pH 

7.4 and water up to 1 ml). The reaction was started by addition of the substrate chlorzoxazone 

and terminated with 3 ml of dichlormethane. 

Determination of the metabolite, 6-hydroxychlorzoxazone, was done by HPLC using LiChrospher 

100 RP-18 endcapped column (Merck, Darmstadt, Germany), (250 x 4.6 i.d.) with mobile 

phase 25% v/v acetonitrile in 0.5% v/v aqueous acetic acid. The flow rate was 1 ml.min - 1 and 

detection at 287 nm according to (8). The HPLC system used was supplied by Shimadzu Class 

VP (Tokyo, Japan) consisting of a LC-10AD quaternary pump, a SIL-10AD autosampler and 

a SPD-10A UV/VIS detector. 

RESULTS AND DISCUSSION 

In all three cases studied, the CYP2E1 activity has been successfully reconstituted. The minipig 

CYP2E1 was fully functional giving realistic values of both maximum velocity of enzymatic 

reaction (V max 

) as well as of the Michaelis constant (K M 

). The highest value of the V max 

was achieved 

with minipig P450 reductase (8.45 ± 1.18 nmol of product/min/nmol P450), the lowest with 

the rat enzyme (6.29 ± 4.46 nmol/min/nmol P450). The lowest value of the Michaelis constant 

indicating the most efficient binding of the substrate was however achieved with the human 

P450 reductase (0.562 ± 0.248 mM). The course of the Michaelis-Menten enzyme kinetics for all 

three P450 reductases are shown in Figure 1. 

The results show that the P450 reductases can be used for reconstitution of the CYP2E1 activity 

regardless on their origin. The differences between V max 

and K M 

values obtained for individual 

P450 reductases are not significant. The reason for this low variability is most probably high 

degree of structural similarity between P450 reductases of different origin in general (3). 

The reasons stressing the importance of the results obtained can be summarized as follows. 

First reason stems from the fact that the studies on the properties of the CYP2E1 enzyme system 

are valuable in all cases as this P450 is known to take part in metabolism of many drugs 

and toxicants as e.g. paracetamol, nitrosamines. The results obtained here show that the origin 

of the reductases does not influence significantly the enzyme activity of this enzyme. Second reason 

is that the results indicate that the CYP2E1 of the minipig is effective in reconstitution systems 

in a similar way as the CYP2E1 enzymes of other species. The results obtained here also 

contribute to the discussion on the suitability of different experimental animals as models in 

experimental pharmacology. Recently, the data obtained have shown that the minipigs or pigs 

seem to be good models for drug metabolism in man (11-13) as well as sources of specialized


Figure 1 

CYP2E1 enzyme kinetics with different P450 reductase enzymes. 

A, minipig liver microsomal P450 reductase; B, rat liver 

microsomal enzyme; C, human recombinant enzyme. Activity 

expressed as nmol product (6-hydroxy chlorzoxazone) formed 

per min per nmol P450. Values of V max 

and of K M 

listed for comparison. 

Figure 1A 

Vmax 

Km 

8450 ± 1180 nmol/min/nmol P450 

0.708 ± 0.274 mM 

Figure 1B 

Vmax 

Km 

6290 ± 4460 nmol/min/nmol P450 

0.728 ± 0.141 mM 

Figure 1C 

Vmax 

Km 

7750 ± 1408 nmol/min/nmol P450 

0.562 ± 0.248 mM 

cells suitable for cell therapy providing the problems with immune response and possible viral 

infection are solved or at least minimalized (14). 

Acknowledgment 

The support from the Medical Faculty of Palacky University of Olomouc to the first author 

(M.B.) is gratefully acknowledged (LF UP grant No 12101105). P.A. thanks for the support 

through the COST B 15.50 project. 

REFERENCES 

1. Horecker BL. Triphosphopyridine nucleotide-cytochrome c reductase in liver. J Biol Chem 1950; 183: 

593-605. 

2. Lu AYH, Coon MJ. Role of hemoprotein P450 in fatty acid (-hydroxylation in a soluble enzyme system 

from liver microsomes. J Biol Chem 1968; 243: 1331-2.

18 


3. Strobel HW, Hodgson AV, Shen S. NADPH cytochrome P450 reductase and its structural and functional 

domains. In: Ortiz de Montellano P.R. editor. Cytochrome P450: Structure, Mechanism, and Biochemistry. 

2 nd ed. New York: Plenum Press; 1995. p.225-44. 

4. Ortiz de Montellano P.R. editor. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2 nd ed. New 

York: Plenum Press; 1995. 

5. Shimada T, Yamazaki H. Cytochrome P450 reconstitution systems. In: Phillips IR, Shephard EA, editors. 

Cytochrome P450 protocols (Meth. Mol. Biol. Vol. 107). Totowa, NJ: Humana Press; 1998. p.85-94. 

6. Wang M, Roberts DL, Paschke R, Shea TM, Masters BSS, Kim JP. Three-dimensional structure of 

NADPH-cytochrome P450 reductase: Prototype for FMN- and FAD- containing enzymes. Proc Natl Acad 

Sci USA 1997; 94: 8411-16 

7. Hubbard PA, Shen AL, Paschke R, Kasper CB, Kim JP. NADPH-cytochrome P450 oxidoreductase. Structural 

basis for hydride and electron transfer. J Biol Chem 2001; 276: 29 163-70. 

8. Lucas D, Berthou F, Girre C, Poitrenaud F, Ménez JF. High-performance liquid chromatographic determination 

of chlorzoxazone in serum: A tool for indirect evaluation of cytochrome P4502E1 activity in 

humans. J Chromatogr B 1993; 622:79-86. 

9. Yasukochi Y, Masters BSS. Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome 

P450) reductase purified by biospecific affinity chromatography. J Biol Chem 1976; 251: 5337-44. 

10. Guengerich FP, Martin MV: Purification of cytochromes P450: Rat and human hepatic forms. In: Phillips 

IR, Shephard EA, editors. Cytochrome P450 protocols (Meth. Mol. Biol. Vol. 107). Totowa, NJ: 

Humana Press; 1998. p.35-54. 

12. Souãek P, Zuber R, Anzenbacherová E, Anzenbacher P, Guengerich FP. Minipig cytochrome P450 enzymes 

have similar properties to human analogs. BMC Pharmacology 2001; 1: 11. 

13. Anzenbacher P, Souãek P, Anzenbacherová E, Gut I, Hrub˘ K, Svoboda Z, Kvûtina J. Presence and activity 

of cytochrome P450 isoforms in minipig liver microsomes. Comparison with human samples. Drug 

Metab. Disposition 1998; 26: 56-9. 

14. Monshouwer M, van’t Klooster GAE, Nijmeijer SM, Witkamp RF, van Miert AS. Characterization of cytochrome 

P450 isoenzymes in primary cultures of pig hepatocytes. Toxicol in Vitro 1998; 12: 715-23. 

15. Horslen SP, Hammel JM, Fristoe LW, Kangas JA, Collier DS, Sudan DL, Langnas AN, Dixon RS, Prentice 

ED, Shaw BW jr, Fox IJ. Extracorporeal liver perfusion using human and pig livers for acute liver failure. 

Transplantation 2000; 70: 1472-8. 

Received: January, 13, 2003 

Accepted: April, 29, 2003


ARBOVIRUSES IN SLOVAKIA 

ELEâKOVÁ, E. 1 , LABUDA, M. 2 , RAJâÁNI J. 1,3 

1 

Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic 

2 

Institute of Zoology, Slovak Academy of Sciences, 845 06 Bratislava, Slovak Republic 

3 

Department of Microbiology and Immunology, Comenius University, Jessenius Faculty of Medicine, 

Faculty Hospital, Martin, Slovak Republic 

Abstract 

Tick-borne encephalitis (TBE) virus is the most important representative of arboviruses in Slovakia. Since 1951, 

when the first epidemic of TBE was described in association with alimentary infection, the topography of natural foci, in 

which the virus circulates, has been repeatedly documented. The reservoirs of TBE are small rodents and insectivores; 

ticks (Ixodes r. spp) transmit the virus and maintain its circulation. The ticks not only behave as TBE virus vectors for wild 

living mammals, but they are also the sites for long-term virus persistence and the source of infection for humans. Typically, 

the endemic natural foci of TBE virus circulation are confined to southern slopes of the Carpathian mountains and 

to the Danube basin. The foci appear most frequently in western parts of the country, but they are also distributed in 

middle and eastern Slovakia. The morbidity of TBE, though actually increasing, ranges from 0.6 to 1.6 per 100.000 inhabitants; 

thus, it still seems relatively moderate, but cannot be neglected. During the last 5 year follow-up (1996-2000), 

the number of TBE patients who acquired the infection by alimentary route was 33; all these patients consumed infected 

raw goat milk or infected sheep cheese. In addition to TBE, another 6 arboviruses were isolated in Slovakia (3 of them 

from mosquitoes), but their medical importance seems less convincing. 

Key words: tick-borne encephalitis virus, Ixodes ricinus ticks, virus transmission, morbidity, natural foci 

INTRODUCTION 

Arboviruses comprise a large group of viruses, which are maintained in nature being transmitted 

to vertebrate hosts by vectors such as ticks and mosquitoes. These vectors replicate as 

well as transmit arboviruses which belong to several families, such Flaviviridae (genus Flavivirus), 

Togaviridae (genus Alphavirus), Bunyaviridae and Reoviridae (Genus Orbivirus). The International 

Catalogue of Arboviruses (1975) lists altogether 359 virus species, out of which 7 have 

been isolated in Slovakia, namely the tick-borne encephalitis (TBE) virus and the West Nile (WN) 

viruses (Flavivirus genus), the Sindbis virus (Alphavirus genus), the ËahyÀa, âalovo and Uukuniemi 

viruses (maily Bunyaviridae) and the Tribeã virus (genus Orbivirus). Out of the above mentioned 

viruses, medically most important is the TBE virus, a serious pathogen causing meningitis, 

encephalitis and paralytic syndrome (1). The TBE virion (Figure 1) contains single stranded 

RNA of plus polarity which is located inside of the capsid built from C protein and surrounded 

by a lipid envelope, containing membrane a (M) protein and a glycoprotein (E). During maturation 

at membrane bound vesicles, which are derived from the endoplasmic reticulum, the M protein 

undergoes N-terminal cleavage. Finally, the mature enveloped particles leave infected cells 

by exocytosis. 

Based on serological (virus neutralization) tests, the Flavivirus genus encompasses 9 virus 

groups, from which 5 consist of viruses transmitted by mosquitoes, 2 groups encounter viruses 

transmitted by ticks and, finally, in 2 groups the vector has not been identified (2). Out of the 6 

serologically distinct viruses which belong to the tick-borne encephalitis serogroup (Table 1), the 

most important members are the Eastern and Western subtypes of TBE virus (3). These two subtypes 

seem closely related, since their structural genes are identical in 86-98% nucleotides (4); 


Assoc. prof. Július Rajãáni, M.D., DSc., Department of Microbiology and Immunology, Comenius University, 

Jessenius Faculty of Medicine, Sklabinská 26, 037 53 Martin 

Phone: ++421 043 4239 038 

e-mail: rajcani@jfmed.uniba.sk

20 


A 

B 

Figure 1. Assembly and maturation of TBE virions in infected cells. The TBE virus nucleocapsid (1A) is surrounded with 

a lipid envelope containing the E and M proteins. During maturation, the M protein undergoes cleavage at its N-terminus. 

The mature particles accumulate within cytoplasmic vesicles (derived from the endoplasmic reticulum) before being 

released from infected cells (1B). 

Table 1. The tick-borne encephalitis (TBE) virus complex. 

Virus type Virus subtype Occurrence of natural foci* 

TBE virus Western (European) Europe from Scandinavia to the Mediterranean 

Sea; from the Atlantic to Red Sea and from 

Finland to Volga river in Kasachstan eastwards 

from Caspian Sea. 

Eastern (Russian spring-summer 

encephalitis viruses) 

Siberia from Ural mountains to the Pacific coast** 

Langat virus 

Powassan virus 

Louping ill virus 

Omsk hemorrhagic fever virus 

Kyasanur forest disease virus 

South East Asia, Philippines 

Canada, USA 

Scotland Ireland Spain Turkey 

Western Siberia 

India 

*see also Figure 2 

**Kamchatka peninsula and the Hokkaido island included 

this also results into a high homology of their corresponding proteins (Table 2). Both TBE viruses 

occur in natural foci, which are widely distributed over the Eurasian continent (Fig. 2), cover 

the whole Europe, Central Asia, and Southern parts of Siberia from Ural mountains towards the 

Pacific coast (3). Within their natural foci, both viruses are transmitted by different vector species, 

namely the Western subtype by Ixodes ricinus ticks, while the Eastern subtype is transmitted 

by Ixodes persulcatus ticks. The essential role of Ixodes ricinus ticks for TBE virus vectors 

has been firmly documented in Slovakia by repeated isolations coming from nymphs as well 

as from mature imagoes (5, 6, 7). Here we report the incidence of Western (European) subtype of 

TBE virus in Slovakia based on our long-term surveillance data. 

MATERIALS AND METHODS 

TBE virus isolation. The tick and organ suspensions (coming from small free living rodents) were 

prepared by tissue homogenization; they were subsequently inoculated into outbred suckling laboratory 

white mice by intracerebral (i.c.) route. Thereafter, the virus isolates were identified by serological 

tests [complement fixation, hemagglutination inhibition (HI)] using a reference antiserum (8, 9). 

Virus titrations. The presence of TBE virus in tick and/or tissue homogenates, in blood and 

in cell culture nutrient fluids was determined in pig kidney (PK) cells grown in plastic 24-well 

plates according to standard procedures (medium BEM with 10% calf serum supplemented with 

antibiotics). Virus titers were read according to developing cytopathic effect and according to 

virus dilutions the TCID 50 

values were calculated.


Table 2. Properties of both TBE virus subtypes. 

European subtype 

Eastern subtype 

Genome Encodes a polyprotein of 3414 aa* Encodes a polyprotein of 3412 aa* 

Encephalitis Focal lesions, lethality 8% 

Vector Ixodes ricinus Ixodes persulcatus 

Distribution Europe, Central Asia Siberia, Eastern Russia 

* amino acid number 

Figure 2. Distribution of the Western (dotted line) and Eastern (solid line) subtypes of TBE virus on the Eurasian continent 

(according to Blaskoviã et al., Bull WHO 1967, 36, pp. 86-94). 

Serological examinations. The presence of specific antibody in various sera coming from small 

rodents, large mammals, goats, sheep and/or captured birds was determined by hemagglutination 

inhibition (HI) tests using a prototype TBE virus antigen prepared from infected mouse brains 

by sucrose-acetone extraction. Four to eight units of antigen were used in each test (9). Before 

use, the sera were adsorbed to goose erythrocytes and delipidized with acetone. 

RESULTS AND DISCUSSION 

Several natural foci of European TBE virus subtype were recognized in Slovakia during the 

last three decades surveillance (Fig.3). Typically, the foci occur in forested areas with an average 

temperature of 8 °C and a yearly waterfall of 800 mm (10). The incidence of Ixodid ticks within 

the foci is relatively high (11). According to their geographical distribution, the Carpathian 

and the Pannonian biotypes can be clearly distinguished (12). The Carpathian foci situated at 

southern slopes of West Carpathian mountains are rich of luxuriant tree forests with herbaceous 

underground. The Pannonian foci, which are situated at lowlands along the Danube river 

basin, are characterized by a highly cultivated landscape separated with small woods consisting 

of oak trees and swampy woods with combined vegetation and a very rich undergrowth. In Slovakia, 

six tick species can be found, Ixodes ricinus, Dermacentor marginatus, D. reticulates, Haemaphysalis 

inermis, H. punctata and H. concinus. From 1964 to 1999 altogether 77,000 ticks 

were collected, pooled and examined for infectious virus presence. Most frequently Ixodes ricinus 

species was idenitified (69,022 inidividuals, i.e. 89.6%), from which 98 TBE virus isolates were 

obtained, an infection rate of 0.14%. However, the TBE virus infection rate of the ticks within 

Carpathian foci may be as high as 2.6%, while the number of infected ticks within the Pannonian 

foci namely at the Danube banks never exceeds 0.1% (13). In general, three conditions favor

22 


Figure 3. The natural foci of TBE virus in Slovakia are situated at the southern slopes of Carpathian mountains (Carpathian 

biotype) and along the Danube river basin (Pannonian biotype). 

Figure 4. Circulation of TBE in a natural focus depicting the accidental infection of man. Note that from domestic animals 

such as goats, the virus may be transmitted via milk (alimentary infection). For details see Gre‰íková M, Kalúzová 

M. Biology of tick-borne encephalitis. Acta Virol. 1997; 41: 115-124. 

to development of natural foci: the virus itself, the presence of the specific vector and the free 

living vertebrate hosts (14). Non-infected ticks acquire the virus during co-feeding with the infected 

ones on the vertebrate host, on small rodents and/or birds (15). Nevertheless, the virus circulation 

is clearly maintained by transmitting the virus from one host to another in association 

with the blood meal. After ingestion, the virus replicates in the gut epithelium cells, within 

the ganglion and in salivary glands of the tick vector and becomes shed by saliva (16). Humans 

have no essential role in virus circulation in natural foci, since they represent the dead end or 

an accidental link (Figure 4).


Table 3. TBE virus infection of Ixodes ricinus ticks collected in regions of Eastern Slovakia in June 1996 and 1997. 

Locality Nymph no Adult no Ticks total Per cent 

Gemerská Poloma 1/29* 3/3 4/32 12.5% 

Miklu‰ovce 1/1 0/8 1/9 11.1% 

NiÏn˘ Medzev n.d. 2/10 2/10 20.0% 

Total 2/30 5/21 7/51 13.7% 

* positive out of total 

Table 4. Occurrence of neutralizing antibodies against TBE virus in small rodents in Slovakia in the years 1964-1996. 

Species Serum sample number Seropositive rate 

Apodemus flavicollis 316/2 105* 15% 

Apodemus sylvaticus 57/626 9.1% 

Apodemus agrarius 6/42 14.3% 

Clethrionomys glareolus 429/2 895 14.8% 

Pitymys subterraneus 13/136 9.6% 

Microtus arvalis 28/344 8.1% 

Other species 6/50 12% 

Total 855/6198 13.8% 


During the last decades, the locations of natural foci have remained surprisingly unchanged; 

however, new foci might have developed and could be discovered based on virus isolation even 

from a relatively small number of collected ticks. For example, in East Slovakia we obtained 

seven TBE isolates from 51 ticks collected at three foci during years 1996-1997; this fact points 

at a relatively high infestation rate of 13.7% and testifies an activity of the foci in question (Table 

3). Thus, the TBE virus may persist within its natural foci for many years. This was demonstrated 

in the Gemerská Poloma locality, where, in addition to the 4 TBE virus isolates coming from 

Ixodes ricinus ticks, another 4 isolates have been obtained from brains of small rodents (Apodemus 

flavicollis, and Clethrionomys glareolus). During the year 1996, thirteen serologically confirmed 

cases of TBE were registered in this area; the seropositive rate among local goats was as 

high as 80%. This example signalizes an exceptionally high infection rate among domestic animals 

and small rodents, who are the hosts for tick vectors. This resulted in accidental infection 

of many human subjects confirming that the natural foci in RoÏÀava district, where the first TBE 

virus focus had been discovered in 1951, have remained active over the period of the last 50 

years. The percentage of infected ticks in the natural foci varies from 0.1% to 5% depending on 

the season and of the focus type. The infected ticks may be transported to long distances by 

migrating birds or to short distances by hunted mammals. Both movements would allow the 

establishment of new foci. About 30% of natural foci in Slovakia were identified according to TBE 

virus isolations from small rodents, namely the yellow necked mouse (Apodemus flavicollis) and 

the bank vole (Clethrionomys glareolus), which not only represent the most frequent virus reservoirs 

but also the most frequent free living mouse species at our territory (17). Therefore, they 

are good indicators reflecting the TBE virus circulation within a given locality. These animals 

reproduce quickly (4-5 months) and are preferred for feeding by nymphs and larvae of the vector 

species. The importance of small rodents for maintaining TBE virus circulation within natural 

foci was also confirmed by following the kinetics of virus neutralizing antibodies (Table 4). In 

addition to ticks and small rodents, the TBE virus was isolated from insectivores (hedgehogs, 

shrews and moles) and birds (wilds ducks, lapwings; (Figure 5). The persistence of TBE in nature 

not only requires a high density of ticks, but also a high population density of vertebrate hosts 

to ensure successful virus transmission (18). Furthermore, ticks feed on large vertebrates such

24 


Figure 5. Isolations of TBE virus from small rodents, insectivores and birds; both biotypes included. The distribution of 

TBE virus alimentary infections is marked by rectangles. Transmission by goat milk was confirmed in RoÏÀava and 

PovaÏská Bystrica districts, while transmission by sheep cheese was described in districts Topoºãany, Luãenec, Gelnica 

and Svidník. 

as goats, sheep, livestock and on large hunted mammals. These contribute indirectly to keep the 

TBE virus circulating. The latter mammals function as indicator hosts rather than as true reservoirs. 

Out of 184 serum samples coming from hunted animals, the seropositive rate ranged from 

3.8 to 27.8% (Table 5). 

The number of TBE cases confirmed by laboratory methods during the last two decades in 

Slovakia ranged from 20 to 90 per year. After an increase in the fifties of 20 th century, some dec-


Table 5. Hemagglutination inhibition (HI) antibodies in large mammals in Western Slovakia from 1997 to 2000. 

Mammal HI antibody titer Per cent 

Wild boar 7/87* 8.0% 

Moufflon 3/79 3.8% 

Deer 5/18 27.8% 


10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

1952 1954 1956 1972 1974 1976 1978 1983 1985 1987 1992 1994 1996 1998 2001 

Figure 6. The TBE morbidity per 100 000 inhabitants during the last four decades in Slovakia (based on cases confirmed by 

laboratory examinations). 

Table 6. Alimentary TBE virus infections in Slovakia during the period from 1951 to 2000. 

Locality District Number of cases Date Vehicle 

RoÏÀava RoÏÀava 271 May 1951 Goat milk 

Závada Topoºãany 12 April 1974 Sheep cheese 

Doln˘ Mo‰tenec PovaÏská Bystrica 4 May 1984 Goat milk 

Dolná Breznica 7 August 1989 Goat milk 

Lednické Rovné 7 September 1993 Goat milk 

Gemerská Poloma RoÏÀava 13 May 1996 Goat milk 

Jaklovce Gelnica 6 May 1998 Sheep cheese 

Ábelová Luãenec 7 May 1999 Sheep cheese 

·ari‰sk˘ ·tiavnik Svidník 4 May 1999 Sheep cheese 

Lednické Rovne PovaÏská Bystrica 3 May 2000 Goat milk 

Table 7. HI antibodies against TBE virus in goats and sheep in alimentary infection areas during years 1996-2000. 

Locality Goats Sheep Peak HI Ab titer 

Gemerská Poloma 42/52* n.d. 1:640 

Jaklovce n.d. 6/246 1:40 

Ábelová 6/64 11/250 1:40 

·ari‰sk˘ ·tiavnik 1/4 6/204 1:80 

Lednické Rovne 1/1 n.d. 1:160 

* positive out of total

26 


Table 8. Arboviruses (other than TBE virus) isolated in Slovakia. 

Virus Host Vector Specific antibodies Pathogenicity Notes 

West Nile Birds Mosquitoes (genus Birds, hunted mammals, Fever, headache, exantema, Isolated from Aedes cantans 

Culex) domesticated mammals lymphadenopathy, arthralgia (25) and from birds 1 (26) alongside 

the Ipeº and Bodva rivers 

Sindbis Birds, frogs, Mosquitoes (genus Birds, hunted mammals, Fever, myositis, headache Isolated from birds 2 (27), 

hamsters Culex) domesticated mammals, man. at Záhorie; isolated from hamsters 3 

(28) at Eastern Slovakia 

and frogs 4 (29) at Záhorie 

ËahyÀa Birds, hair, hedghog, Mosquitoes Aedes Birds, hunted „flu“-like syndrome Isolated from Aedes vexans 

deer, fox, mammals, man or caspius (30) at East Slovakia 

and from human blood (31) 

âalovo Birds, livestock, Mosquitoes Domestic animals, man, Mild disease with fever Isolated from Anopheles mac. 

horses, swine hunted mammals, water birds (32) at the Danube basin 

Uukuniemi Birds, small rodents Ixodid ticks Birds, man Not determined Isolated from Ixodes ricinus (33) 

and from mice 5 [34] at Topoºãianky 

district 

Tribeã Small rodents Ixodid and Man at low rate Mild meningitis 6 Isolated from ticks 7 and small 

Haemaphysalis ticks rodents in Tribeã mountains (35) 

1 

Vanellus v., Streptopalia t., Tringa ochropus; 2 Acrocephalus s.; 3 Cricetusc c.; 4 Rana r.; 

5 

Apodemus flavicollis; 

6 

Experimentally confirmed in monkeys using the related Kemerovo virus (36); 7 Ixodes ricinus


line was observed in the seventies. However, from 1994 the number of clinically apparent cases 

began rising again, so that the morbidity nowadays exceeds 1/100 000 (Figure 6). The majority 

of cases has remained associated with the activity of ticks in May and/or June. TBE may be 

regarded for an occupational disease of agricultural and forest workers. Tourists may become 

affected when coming to natural foci. The highest morbidity is observed in Western Slovakia due 

to a higher density of foci in this part of the country. 

During the first well documented outbreak in RoÏÀava in 1951, the virus was found to infect 

children by alimentary route after ingestion of raw goat milk (19). Alimentary infections appear 

in small epidemics with a family confined occurrence (20, 21, 22, 23). All such microepidemics 

registered in Slovakia were either due to ingestion of goat milk or sheep cheese (Table 6). Figure 

5 also shows the areas, where alimentary infections occurred; these locations, of course, only 

partially overlap with the distribution of free living rodents, i.e. only in a few natural foci the TBE 

virus gets frequently transmitted to domesticated milk giving mammals. As already mentioned, 

the seropositive rate of HI antibodies in goats of a single focus has reached the alarming value 

of 80%. Experimentally infected goats and/or sheep would develop viraemia followed by excretion 

of the virus to milk [24]. Thereafter, alimentary transmission occurs, if the virus was not properly 

inactivated by pasteurization or if infected raw milk has been ingested. During the period 

of 1996-2000 altogether 33 patients from 5 districts were enrolled to Slovakian hospitals who 

had TBE and a history of alimentary infection by goat milk or sheep cheese. Though the virus 

could not be always isolated from the milk product in question, the presence of high HI antibody 

titers in the corresponding goats and/or sheep was obvious (Table 7). 

Despite of its moderate morbidity and lower lethality (Table 2, Figure 6), the European type 

TBE still represents a permanent risk, especially in the natural foci associated with an endemic 

occurrence of the disease. Epidemiological surveyllance of the documented natural foci and further 

active search for potential new ones is based on the history of TBE cases. Such investigations 

are still relevant. For protection of subjects frequently visiting the natural foci, vaccination 

is indicated. Immunoprophylactic vaccination seems inevitable for subjects who are professionally 

exposed to tick bite when working within natural foci. Relevant information about the territory 

of foci and of the vaccine itself should be available for tourists and even for wide public. 

Though the medical importance of the rest of arboviruses isolated in Slovakia is less striking 

than that of TBE virus, non of them can be fully neglected (Table 8). 

REFERENCES 

1. Westaway EG, Brinton MA, Gaidamovich SY, Horzinek MC, Igarashi A, Kaariainen L, Lvov DK, Porterfield JS, Russell 

PK, Trent DW. Flaviviridae. Intervirology 1985; 24 : 183-192. 

2. Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Mayo MA, Summers MD. (ed.) Virus Taxonomy. Classification 

and Nomenclature of Viruses. Wien-New York, Springer Verlag, 1995. 

3. Gre‰íková M, Calisher CH. Tick-borne encephalitis. In: The Arboviruses: Epidemiology and Ecology (T. P. Monath, 

ed.) CRC Press Inc., Boca Raton, Florida 1988: 177-202. 

4. Mandl CW, Heinz FX, Kunz C. Sequence of the structural proteins of the tick-borne encephalitis virus (western subtype) 

and comparative analysis with other flaviviruses. Virology 1988; 166: 197-205. 

5. Gre‰íková M, Nosek J. Isolation of tick-borne encephalitis virus from Ixodes ricinus ticks in the Tribeã region. Bull 

Wld Hlth Org. 1967; Suppl 1: 67-71. 

6. Gre‰íková M, Sekeyová M. Characteristics of some tick-borne encephalitis virus strains isolated in Slovakia. Acta 

Virol 1980; 24: 72-75. 

7. Gre‰íková M, Palanová A, Potheová A, Teplan J, Sekeyová M, Kohútová V. Contribution to the detection of a natural 

focus of tick-borne encephalitis in the south of middle Slovakia. Bratisl lek Listy 1983; 80 (4): 385-512. 

8. Casals J, In: Maramorosh K, Koprowski H. (Eds.). Methods in Virology. New York – London, Academic Press 1967; 

3: 163-181. 

9. Clarke DH, Casals J. Techniques for haemagglutination and haemagglutination-inhibition with arthropod-borne 

viruses. Am J Trop Med Hyg 1958; 7: 561-573. 

10. Labuda M, KoÏuch O, Lys˘ J. Tick-borne encephalitis natural foci in Slovakia: ticks, rodents, and ...goats. In: Tickborne 

Encephalitis and Lyme Borreliosis, Potsdam Symposia (J. Suess and O. Kahl, eds.) Pabst Science Publishers, 

Lengerich 1997: 34-46. 

11. Nuttall PA, Labuda M. Tick-borne encephalitis subgroup. In: Ecological Dynamics of Tick-borne Zoonoses (D. E. 

Sonenshine and T. N. Mather, eds.) Oxford University Press, New York, Oxford 1994: 351-391.

28 


12. Gre‰íková M. Studies on tick-borne arboviruses isolated in Central Europe. Biological works SAV, Bratislava 1972; 

18: 1-111. 

13. Gre‰íková M, Nosek J. Arboviruses isolated from ticks in Central Europe. Biologia 1982; 37: 755-763. 

14. Pavlovskij EN. Kle‰ãi i kle‰ãevoj encefalit. Parazitologija Daºnego Vostoka Medgiz. Moskva 1947: 212. 

15. Labuda M, Danielova, V, Jones, LD, Nutall, PA: Amplification of tick-borne encephalitis virus infection during co/feeding 

of ticks. Med Vet Entomol 1993; 7; 339-342. 

16. Rajãáni J, Nosek J, KoÏuch O., Waltinger H.: Reaction of the host to the tick bite: Distribution of tick borne encephalitis 

virus in sucking ticks. Zbl Bakt Hyg I, Abt. Orig. A 1976; 236; 1-9. 

17. KoÏuch O, Labuda M, Lys˘ J, Weismann P, Krippel E. Longitudinal study of natural foci of Central European encephalitis 

virus in West Slovakia. Acta Virol 1990; 34: 537-544. 

18. Labuda M, Austyn JM, Îuffová E, KoÏuch O, Nuttall PA. Importance of localised skin infection in tick-borne encephalitis 

virus transmission. Virology 1996; 219: 357-366. 

19. Bla‰koviã D. An epidemic of encephalitis in the natural infective focus in RoÏÀava. Vydavateºstvo SAV, Bratislava; 

1954. 

20. Gre‰íková M, Sekeyová M, Stúpalová S, Neãas S. Sheep milk-borne epidemic of tick-borne encephalitis in Slovakia. 

Intervirology 1975; 5: 57-61. 

21. Îaludko J, Vrbová O, Hachlincová R, Kohl I, Hubálek Z, Jufiicová Z, KoÏuch O, Eleãková E, Labuda M. Cases of family 

epidemics of tick-borne encephalitis in the central part of PovaÏie. Bratisl lek Listy 1994; 95: 523-526. 

22. Kohl I, KoÏuch O, Eleãková E, Labuda M, Îaludko J. Family outbreak of alimentary tick-borne encephalitis in Slovakia 

associated with a natural focus of infection. Europ J Epidemiol. 1996; 12: 373-375. 

23. Kohl I, Gre‰íková M, Kohútová V, Îaludko J, Sekeyová M. Investigations on a natural focus of tick-borne encephalitis 

in the PovaÏská Bystrica district. Biologia (Bratislava) 1989; 44: 267-273. 

24. Gre‰íková M. Recovery of the tick-borne encephalitis virus from the blood and milk of subcutaneously infected sheep. 

Acta Virol 1958; 2: 113-119. 

25. Labuda M, KoÏuch O, Gre‰íková M. Isolation of West Nile virus from Aedes cantans mosquitoes in West Slovakia. 

Acta virol 1974; 18: 429-433. 

26. Gre‰íková M, Sekeyová M, Prazniaková E. Isolation and identification of group B arboviruses from the blood of birds 

captured in Czechoslovakia. Acta Virol 1975; 19: 162-164. 

27. Ernek E, KoÏuch O, Nosek J, Gre‰íková M, Sekeyová M. Isolation of Sindbis virus from the reed warbler (Acrocephalus 

scirpaceus) in Slovakia. Acta virol. 1973; 17: 359-361. 

28. Gre‰íková M, Sekeyová M, Batíková M, Bieliková V. Isolation of Sindbis virus from organs of a hamster in East Slovakia. 

1. Internationales Arbeitskolloquium ıber Naturherde von Infektionskrankheiten in 17-19. april 1973, Illmitz 

und Graz 1973, p. 112-113. 

29. KoÏuch O, Labuda M, Nosek J. Isolation of Sindbis virus from the frog Rana ridibunda. Acta Virol 1978; 22: 78. 

30. Bárdo‰ V, Danielová V. Virus ËahyÀa – virus isolated from mosquitoes in Czechoslovakia. J Hyg Epid Microbiol 

Immunol 1959; 3: 264-276. 

31. ·imková A, Sluka F. Isolation of ËahyÀa virus from the blood of a case of influenza – like disease. Acta Virol 1973; 

17: 94. 

32. Bárdo‰ V, Danielová V. Study on the relationship of ËahyÀa virus – Aedes vexans under natural conditions. âs epid 

Mikrobiol Imunol 1961; 10: 389-395. 

33. KoÏuch O, Gre‰íková M, Nosek J. Poteplí – Uukuniemi virus isolated from Ixodes ricinus ticks in Slovakia. Acta Virol 

1968; 12: 475. 

34. KoÏuch O, Rajãáni J, Sekeyová M, Nosek J. Uukuniemi virus in small rodents. Acta Virol 1970; 14: 163-166. 

35. Gre‰íková M, KoÏuch O, Ernek E, Nosek J. „Tribeã“ a newly isolated virus from tick Ixodes ricinus and small rodents 

In. B. Rosick˘ and K. Heyberger (ed.): Theoretical questions of natural foci of diseases. Proceeding of a symposium. 

Czechoslovak Acad. Sci. Prague. 1965; p. 439. 

36. Líbiková H, Tesafiová J, Rajãáni J. Experimental infection of monkeys with Kemerovo virus. Acta Virol 1970; 14; 64- 

69. 

Received: May, 29, 2003 



PREVALENCE OF PROTHROMBIN MUTATION GENE (G→A 20210) 

IN THROMBOPHILIC PATIENTS 

JURAJ CHUDEJ 1 , STANISLAVA KONEâNÁ 1 , IVANA PLAME≈OVÁ 1 , JELA IVANKOVÁ 1 , 

JAN HUDEâEK 1 , JÁN STA·KO 1 , RUDOLF PULLMANN 2 , VLADIMÍR MELU·2 , PETER KUBISZ 1 

1 

Department of Hematology and Blood Transfusion, National Centre of Hemostasis and Thrombosis, 

Comenius University, Jessenius Faculty of Medicine, Faculty Hospital, Martin, Slovak Republic 

2 

Department of Clinical Biochemistry, Comenius University, Jessenius Faculty of Medicine, Faculty Hospital, 

Martin, Slovak Republic 

Abstract 

Pathogenesis of venous thrombosis is multifactorial. In more than 40% of patients it is a familiar affliction, based on different 

congenital defects of haemostasis e.g. PC, PS, AT III deficiency or a resistance to activated protein C (APC-R), or 

recently discovered variant of prothrombin 20210 A. 

In this study we investigated the prevalence of the prothrombin 20210A mutation in our population of patients with 

thrombophilic state. We have detected higher prevalence of this gene mutation (8,6 %) in a group of our so far examined 

patients than it is in other neighbouring countries (Poland, Italy, England, Sweden, France). 

Key words: prothrombin gene mutation (G→A 20210), thrombophilia, DNA analysis 

INTRODUCTION 

Mutation of prothrombin gene that leads to variant PT 20210 G→A was described in 1996 by Poort 

et al.(1). PT 20210 mutation connected with threefold higher risk of thrombosis occurs in the 3rd 

untranslated part of the gene on position 20210A (PT 20210 G→A) (1,2). This defect is transmitted as 

an autosomal dominant disorder. Mutation was found in 18% of patients in a study of selected members 

of families with positive anamnesis of venous thrombosis (1). Presence of this mutation was discovered 

in 20% of patients with cerebral venous thrombosis. The gene increases a risk of thrombosis 

onset 10.2 times (3). Role of prothrombin allele 20210A in pathogenesis of arterial thrombosis is still 

a subject of discussion (4,5). In another study, 87% of patients with PT mutation had level of prothrombin 

more than 115 % of a normal level, with 2.1 fold increase in risk of thrombosis (6,7). In the 

past it was thought that assessment of prothrombin level would be a good screening test for this mutation 

(7,8). Nevertheless, recent studies have proved just a minimal relationship between prothrombin 

plasma levels and the presence of this mutation (9). Prevalence of the variant PT 20210 G→A is different 

in various populations (10,11). Similar to prevalence of FV Leiden, occurrence of PT 20210 G→ A 

mutation is higher in European population and only very rare in African and Asian populations. The 

alleles seem to be rare among native Americans, black Africans, Amazonian Indians, Asians, Indians 

and Inuits (10,11). Rosendaal et al. have discovered 3% prevalence among inhabitants of southern 

Europe and 1.7% in the population of northern Europe (10,11). In northern Europe PT 

20210G→A occurs in 2% of thromboses. In Catalonia (Spain) where the prevalence of this anomaly 

is almost 6.5%, PT 20210 G→A is a cause of more than 6% of thromboses (12). In countries of the 

southern Europe variant prothrombin is more frequent cause of a thrombophilic state than FV Leiden 

(9,11). Very obvious is an interaction with other risk factors e.g. hormonal therapy with oral contraceptives 

that elevate risk of thrombosis 150 times (3, 13, 14 ). 

METHODS 

For DNA analysis of prothrombin 20210 G→A gene mutation sample of 4 ml of venous blood is 

taken into a special test-tube with content of 400 µl 0.5 M EDTA. Lysis of erythrocytes is done by 


Prof. Peter Kubisz,M.D., DSc., Department of Hematology and Blood Transfusion, 

National Centre of Hemostasis and Thrombosis, Comenius University, Jessenius Faculty of Medicine 

and Faculty Hospital, Kollárova 2, 036 59 Martin, Slovak Republic 

Phone: ++421 43 413 3308, 4203 233, Fax: ++421 43 4132 061 

e-mail: kubisz@jfmed.uniba.sk

30 


Figure 1 

Lane M: molecular weight marker 

Lane 1: FII allele 20210 cleaved PCR product (345 bp → 322 bp + 23 

bp* by Hind III restriction enzyme) 

Lane 2: Heterozygous 20210 G/A genotype (345 bp, 322 bp, 23 bp*) 

Lane 3: Homozygous 20210 A/A genotype (322 bp, 23 bp*) 

*: 23 bp fragment is too small and migrating out of agarose gel 

(i.e. 23 bp fragment isn’t present at the picture) 

Lane 4: Homozygous 20210 G/G genotype (healthy individual ‘– 345 bp) 

– 345 bp 

– 322 bp 

M 1 2 3 4 

– 23 bp 

a lysing hypotonic solution (0,155 M NH 4 

Cl + 0,17 M Tris-HCl, pH = 7.6, 9 : 1). It is mixed with blood 

in the ratio 6 : 1.This is followed by an incubation in water thermostat at the temperature of 37 °C. 

Leucocytes are separated from the sediment by centrifugation (4.000 g, 10 min, 25 °C) and then washed 

by 0,15 M NaCl solution. After another centrifugation the sediment consisting of leucocytes is dissolved 

in a high-concentration buffer solution TE (100 mM Tris-HCl, 40 mM EDTA, pH = 8) and this 

volume is mixed with a lysing mixture (0.2% SDS, 1M NaCl, 40 mM EDTA, 100mM Tris-HCl, pH = 8) 

in 1:1 ratio. Cellular debris is removed by adding an equivalent amount of the mixture phenol : chloroform 

(3 : 1). Then the mixture is divided by a centrifugation into few phases, the top pure one contains 

DNA. The phase containing DNA is rewashed by an equivalent amount of chloroform : izoamylalcohol 

(24 : 1) and then centrifugated again. The top layer, rich in DNA is afterwards mixed with an equivalent 

amount of the mixture of izopropylalcohol : NH 4 

Ac (10 : 1). DNA in this mixture coagulates into 

a form of white clouds, that are transferred into a microtest-tube. DNA has to be purified from salts by 

70 % alcohol. After centrifugation (10.000 g, 10 min, 25 °C) the DNA sediment is dried in a vacuum and 

dissolved in a buffer solution TE (10 mM Tris-HCl, 1 mM EDTA). DNA specimen processed in this way 

is suitable for PCR genetic analysis. Presence of prothrombin gene mutation 20210 G→A is assessed 

by a polymerase chain reaction (PCR). Method is based on enzyme amplification of selected part of DNA 

in vitro. A subsequent effect of a restrictive endonuclease (in our case Hind III) cleaves selected DNA 

into fragments, which size and amount depends on the presence of the mutation. Fragments of DNA 

are visualized by an agarose gel electrophoresis. The method enables to distinguish a healthy individual 

from a heterozygous or homozygous carrier of prothrombin gene mutation (1; Figure1). 

RESULTS 

In our study, we examined 2046 patients with thromboembolic disease in their clinical history 

(deep venous thrombosis, pulmonary embolism). 

Presence of prothrombin 20210A gene mutation was detected in 176 patients (8,6%) with VTE. 

Homozygous carriers represent 1.8% and heterozygotes create 98.2% of this count (Table1).


Table 1. Prevalence of prothrombin variant 20210A in the thrombophilia patients. 

Mutation 20210 G→A Heterozygotes G/A Homozygotes A/A 

8.6% (176) 98.2% (175) 1.8% (1) 

DISCUSSION 

From comparison with known results of the prevalence of prothrombin gene mutation in 

thrombophilic patients in different countries (10, 11), it is obvious that prevalence of the mutation 

of this hereditary thrombophilia in Slovakia is much higher than European average. 

Prothrombin gene mutation 20210 G→A, can be asymptomatic. More often we can observe 

maniphest trombophilia e.g. deep venous thrombosis, pulmonary embolism, even in young individuals 

(11, 15 ). The prevalence of prothrombin mutation in Europe varies from 1% to 6,5% (except 

of Catalonia) (10, 11, 12). In our study we have observed higher prevalence of prothrombin 

20210G→A gene mutation in comparison with European average prevalence. It is therefore necessary 

to identify a degree of the risk, in order to prevent thrombosis (prophylaxis in stress situations 

in identified defects). It is important to search for endangered individuals and define situations 

that are potentially thrombogenic so that we could decide for an appropriate therapy to prevent 

recidivation and indicate a necessary length of therapy to ensure secondary prevention. It is 

important, as well, to examine members of the affected family (direct relatives of the patient) so 

that in stress situations (immobilisation, surgery, pregnancy, puerperium) we can use an appropriate 

prophylaxis. 

Our results should be considered to have a preliminary value because of the limited number 

patients in the study and their confirmation requires further research. 

REFERENCES 

1. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3’-untranslated region of the 

prothrombin gene is associated with elevated plasma prothrombin levels and increase in venous thrombosis. Blood, 

1996; 88: 3698-3703. 

2. Galajda P, Hulíková M, Kubisz, P. Trombofilné stavy. Medicínsky Monitor, 2000; 4: 16-20. 

3. Frenkel EP, Buk RL. Prothrombin G20210A gene mutation, heparin cofactor II defects, primary thrombocytemia and 

thrombohemorrhagic manifestation. Seminars in thrombosis and haemostasis, 1999; 25, 4: 376-385. 

4. Bertina RM. The prothrombin 20210 G to A variation and thrombosis. Curr-Opin-Hematol, 1998; 5 (5) : 339-342. 

5. Vargas M, Soto I, Pinto CR, Urgelles MF, Batalla A, Rodriguez-Reguero J, Cortina A, Alvarez V, Coto E. The prothrombin 

20210A allele and the factor V Leiden are associated with venous thrombosis but not with also early coronary 

artery disease. Blood-Coagul-Fibrinolysis, 1999; 10 (1): 39-41. 

6. Bertina RM. Molecular risk factors for thrombosis. Thromb Haemost, 1999; 82(2): 601-609. 

7. Eikelboom JW, Ivey L, Baker RT. Familial thrombophilia and the prothrombin 20210A mutation associated with increased 

thrombin generation and unusual thrombosis. Bloodcoagulation-fibrinolysis, 1999; 10 (1): 1-5. 

8. Simoni P, Tormene D, Manfrin D, Gavasso S, Luni S, Stocco D, Girolami A. Prothrombin antigen levels in symptomatic 

and asymptomatic carriers of the 20210A prothrombin variant. Br-J-Haematol, 1998; 103(4): 1045-1050. 

9. Chamouard P, Pencreach E, Maloisel F, Grunebaum L, Meyer A, Gaub MP, Goetz J, Baumann R, Uring-Lambert B, 

Levy S, Dufour P, Hauptmann G, Oudet P. Frequent factor II G20210A mutation in idiopathic portal vein thrombosis. 

Gastroenterology, 1999; 116: 144-148. 

10. Rosendaal FR, et al. Geografic distribution of the 20210 G to A prothrombin variant. Thromb Haemost, 1998; 79: 706 

11. Rosendaal FR. Risk factors for venous thrombotic disease. Thromb Haemost, 1999; 82 (2): 610-619. 

12. Souto JC, Coll I, Llobet D, del-Rio-E, Oliver A, Mateo J, Borell M, Fontcuberta J. The prothrombin 20210A allele is 

the most prevalent genetic risk factor for venous thrombembolism in the Spanish population. Thromb Haemost, 

1998; 80 (3): 366-369. 

13. van Krimpen J, Leebeek FW, Dippel DW, Gomez Garcia E. Prothrombin gene variant (G20210A) in a patient with 

cerebral venous sinus thrombosis. Clin Neurol Neurosurg, 1999; 101(1): 53-55. 

14. Eichinger S, Minar E, Hirschl M, Bialonczyk C, Stain M, Mannhalter C, Stumpflen A, Schneider b, Lechner K, Kyrle 

PA. The risk of early recurrent venous thromboembolism after oral anticoagulant therapy in patient with the 

G20210A transition in the prothrombin gene. Thromb Haemost, 1999; 81 (1): 14-17. 

15. De Stephano V, Chiusolo P, Paciaroni K, Casorelli I, Rossi E, Molinari M, Servidei S, Tonali PA, Leone G. Prothrombin 

G20210A mutant genotype is a risk factor for cerebrovascular ischemic disease in young patients. Blood, 1998; 

91, 3562-3565. 

Received: April, 14, 2003 


32 


CENTRAL VERSUS INTRAPARENCHYMAL ARTERIES OF KIDNEY: 

COMPARISON OF DOPPLER PARAMETERS UNDER THE PHYSIOLOGICAL 

CONDITIONS IN NEWBORNS 

MIROSLAV STAVùL 1 , HANA KOLLAROVSZKA 1 , ZUZANA STRECHOVÁ 1 , HUBERT POLÁâEK 2 , 

MIRKO ZIBOLEN 1 

1 

Clinic of Neonatology, Comenius University, Jessenius Faculty of Medicine, Faculty Hospital, Martin, Slovak Republic 

2 

Department of Radiology, Comenius University, Jessenius Faculty of Medicine, Faculty Hospital, Martin, 

Slovak Republic 

Abstract 

The objective of this research was to evaluate renal blood flow in healthy newborns by means of Doppler ultrasonography. 

A total number of 40 newborns were examined. The blood flow in central renal arteries versus intraparenchymal 

arteries was compared. Maximum systolic velocity (V max), end-diastolic velocity (V ed), mean flow velocity (V mean), 

resistive index (RI) and pulsatility index (PI) were assessed. Values of all the named parameters evaluated in central renal 

arteries are significantly higher than the values of the same parameters evaluated in the intraparenchymal arteries. Measured 

values could be considered as physiological ones in this age group. 

Key words: Doppler, newborn, kidney, resistive index 

INTRODUCTION 

Renal sonographical screening at the neonatal units allows an early detection of any developmental 

abnormalities and acute states in the perinatal period (1). Doppler measurement methods 

broaden the conventional sonography and allow to evaluate not only the structure of kidneys 

but also renal haemodynamics. It is also possible to perform it even in the prenatal period 

(2). A detection of blood flow changes, which can precede the structural ones, permits us in some 

cases to establish the diagnosis before a conventional sonography or prior to the time when clinical 

signs become apparent. It is useful with different renal pathological states, e.g. acute tubular 

necrosis in perinatal asphyxia (3, 4) or for distinguishing obstructed from non-obstructed 

hydronephrosis (5, 7). It informs not only about the process of disease and its prognosis but also 

allows the start for an early treatment. 

The problem of renal Doppler measurements in neonatal period and infancy is a minimum of 

researches devoted to this area (8, 9, 10, 11). As a consequence there are no generally accepted 

normative values of Doppler parameters in this age group, especially in the early postpartal period. 

There were healthy neonates studied in the present research. The aim of this research was 

to establish physiological normative values of selected Doppler parameters of renal circulation. 

MATERIAL AND METHODS 

There were 40 healthy neonates (21 girls and 19 boys) from physiological pregnancies with 

negative perinatal history enrolled in this study. All of them were born in term (average of 40.2 

± 1.1 gestational weeks). All had an appropriate weight for gestational age, following the criteria 

of British Child Growth Fundation (average of 3519 ± 412 g). Values of Apgar score in 5 th and 

10 th minute were 8 or more, there were no signs of hypoxia. The examination was performed on 

4 th –5 th day of their life (average of 84 ± 10 hours of life). There were no clinical signs indicating 


Miroslav Stavûl, M. D., Clinic of Neonatology, Comenius University, Jessenius Faculty of Medicine and Faculty Hospital, 

Kollárova 2, 036 59 Martin, Slovak Republic 

Phone: ++421 43 4203 641, Fax: ++ 421 43 4222 678 

e-mail: stavel@lefa.sk


Table 1. Comparison of Doppler parameters of central renal versus intraparenchymal arteries 

AR IP P value 

V max (cm/s) 40.8 ± 13.7 7.0 ± 1.0

34 


Figure 1. Comparison of resistive index of central renal 

versus intraparenchymal arteries (AR – central renal arteries, 

IP – intraparenchymal arteries, RI – resistive index). 

Figure 2. Comparison of pulsatility index of central renal 

versus intraparenchymal arteries (AR – central renal arteries, 

IP – intraparenchymal arteries, PI – pulsatility index). 

Analogous comparison of all listed parameters in this age group has not been published yet 

in available sources. 

Decline of RI from last trimester of pregnancy to 6 th month of life was presented by Andriani 

et al (11). In neonatal period they mention the values of RI between 0.57-0.90. Only renal artery 

was assessed. Values of RI are in concordance with our results on renal artery. 

Lin and Cher (9) in their research on healthy children of age between 0-13 years pointed out 

a continuous decline of RI along with age. Values mentioned in the age group of 0-3 months in 

renal arteries (RI = 0.69 ± 0.10, PI = 1.22 ± 0.30) are lower than values recognized in presented 

research that could be related with age inhomogeneity of their group. 

Evaluation of renal circulation from late foetal period until one year was the aim of the research 

published by Veille et al (8). They reported a significant increase of the renal artery diameter, 

time/velocity integral, peak flow velocity, mean flow velocity and absolute renal flow along 

with age. A decline of systolic/diastolic velocity ratio was noticed in this period. Renal blood flow 

related to body weight did not change significantly along with age. All measurements were performed 

on renal artery and on a small group (19 children). 

Kuzmic et al (13) compared RI of different children’s age groups in their researches. They rated 

RI on interlobar and arcuate arteries. They recognized significantly higher RI (0.705 ± 0.018) in 

the youngest age group than in the older ones (0.605 ± 0.029 and 0.0604 ± 0.035 respectively). 

The age groups were divided into two groups of children aged between 2-6,then 6-16 years and 

adults, therefore the interpretation of neonatal values according to this research is limited. 

Comparison of parameters on different levels of renal vascular tree was assessed by Rivolta 

et al (14). They performed their measurements on the renal artery, interlobar arteries and cortical 

arteries. They recognized significantly lower values of RI in cortical arteries in comparison 

with interlobar and renal arteries. Examined group were adults, however, their results concord 

with our results. Thesis of renal vascular resistance decline towards peripheral arteries under 

physiological conditions is probably applicable in all age groups. 

Presented research follows preliminary Zibolen’s study (6, 7), who has assessed 14 newborns 

aged 0-1 months. The significant difference of RI, PI, Vsyst and V ed between renal and intraparenchymal 

arteries is in concordance with presented up-to-date measurements, although 

absolute values are different. We attribute it to different age of examined babies and different file 

size. Limited equipment facilities in the time of their study have influenced the results too. 

In conclusion, we want to point out the specificity of renal circulation in neonatal period. 

However, these preliminary results already indicate that physiological values of elder age groups 

have limited use in this period. Big variety of published results in the past is due to differences 

in the location of the measurement. It is desirable to establish physiological values of selected 

Doppler parameters in this age group.


REFERENCES 

1. âerveÀová O, âernianska A, Chocholová M, Mikloviãová D, Kizeková Z, Büchler P, VaÀatka R, Polák V, Wasserman 

O. „Rub a líc” 10 roãného skríningu ob‰trukãn˘ch uropatií. In: Benedeková et al. Klinická pediatria I. Bratislava: Univerzita 

Komenského; 2002. p. 29-32. 

2. Vi‰Àovsk˘ J. Vy‰etrenie cirkulácie v pôrodníctve. Martin: Osveta; 2002. 

3. Luciano R, Gallini F, Romagnoli C, Papacci P, Tortorolo G. Doppler evaluation of renal blood flow velocity as a predictive 

index of acute renal failure in perinatal asphyxia. Eur J Pediatr 1998; 157 (8): 656-60. 

4. Streitman K, Tóth A, Horváth I, Tálosi G. Renal injury in perinatal hypoxia: ultrasonography and changes in renal 

function. Eur J Pediatr 2001; 160 (8): 473-7. 

5. Svitac J, Zibolen M, Kliment J, Buchanec J. Renal Doppler ultrasonography in infants with hydronephrosis. Int Urol 

Nephrol 2001; 33 (3): 431-3. 

6. Zibolen M. Colour duplex Doppler sonographic comparison of blood flow in renal and intraparenchymal renal arteries 

in healthy neonates. Eur J Pediatr 1994; 153 (8): 614. 

7. Zibolen M. Dopplerovská sonografia obliãiek plodov, novorodencov a detí. Martin: Flipo; 2000. 

8. Veille JC, McNeil S, Hanson R, Smith N. Renal Hemodynamics: Longitudinal study from the late fetal life to one year 

of age. J Matern Fetal Investig 1998; 8 (1): 6-10. 

9. Lin GJ, Cher T Renal vascular resistance in normal children – a color Doppler study Pediatr Nephrol 1997; 11 (2): 

182-5. 

10. Scholbach T. Color Doppler sonographic determination of renal blood flow in healthy children. J Ultrasound Med 

1999; 18: 559-64. 

11. Andriani G, Persico A, Tursini S, Ballone E, Cirotti D, Lelli Chiesa P. The renal-resistive index from the last 3 months 

of pregnancy to 6 months old. BJU Int 2001; 87 (6): 562-4. 

12. Jurko A jr. Vybrané echokardiografické parametre novorodencov a dojãiat. Martin: Flipo; 2000. 

13. Kuzmic AC, Brkljacic B, Ivankovic D, Galesic K. Doppler sonographic renal resistance index in healthy children. Eur 

Radiol 2000; 10 (10): 1644-8. 

14. Rivolta R, Cardinale L, Lovaria A, Di Palo FQ. Variability of renal echo-Doppler measurements in healthy adults. 

J Nephrol 2000; 13 (2): 110-5. 

This study was supported by Grant VEGA No. 1/0044/03. 

Received: January, 10, 2003 

Accepted: March, 20, 2003

MAKETA po korektœre 2003

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?